Glycoconjugation process

ABSTRACT

The present disclosure relates generally to methods of preparing glycoconjugates containing a saccharide conjugated to a carrier protein by use of stable nitroxyl radical related agent/oxidant as an oxidizing agent, to immunogenic compositions comprising such glycoconjugates, and to methods for the use of such glycoconjugates and immunogenic compositions.

FIELD

The present disclosure relates generally to methods of preparingglycoconjugates containing a saccharide conjugated to a carrier proteinby use of TEMPO/NCS as an oxidizing agent, to immunogenic compositionscomprising such glycoconjugates, and to methods for the use of suchglycoconjugates and immunogenic compositions. The present disclosurealso relates to methods of preparing glycoconjugates containing asaccharide conjugated to a carrier protein, by the use of stablenitroxyl or nitroxide radicals such as piperidine-N-oxy orpyrrolidine-N-oxy compounds in the presence of an oxidant to selectivelyoxidize primary hydroxyls of the said saccharide, to immunogeniccompositions comprising such glycoconjugates, and to methods for the useof such glycoconjugates and immunogenic compositions.

BACKGROUND

Polysaccharide protein conjugate vaccines are made usingpolysaccharides, generally from the surface coat of bacteria, linked toprotein carriers. The chemical bonding of the polysaccharide and proteincarrier induces an immune response against bacteria displaying thepolysaccharide contained within the vaccine on their surface, thuspreventing disease. Accordingly, vaccination using polysaccharides frompathogenic bacteria is a potential strategy for boosting host immunity.The polysaccharides that cover bacteria vary greatly, even within asingle species of bacteria. For example, in Streptococcus pneumoniae (aleading cause of meningitis, pneumonia, and severe invasive disease ininfants and young children throughout the world) there are more than 90different serotypes due to variation in the bacterial polysaccharidecoat. Therefore, polysaccharide vaccines often consist of a panel ofpolysaccharides to increase protection.

Although polysaccharides are immunogenic on their own, conjugation ofpolysaccharides to protein carriers has been used to improveimmunogenicity. The carrier protein can be either a related proteinantigen from the target pathogen, boosting the specific immune responseto that pathogen, or a generally immunogenic protein that serves more asan adjuvant or general immune response stimulant.

Multivalent pneumococcal polysaccharide-protein conjugate vaccines havebeen licensed for many years and have proved valuable in preventingpneumococcal disease in infants and have recently been recommended foradults.

SUMMARY

In one aspect, the present disclosure provides a method of making aglycoconjugate comprising a saccharide conjugated to a carrier protein,comprising the steps of: a) reacting a saccharide with2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide(NCS) in an aqueous solvent to produce an activated saccharide; and b)reacting the activated saccharide with a carrier protein comprising oneor more amine groups. In a further aspect, the degree of oxidation ofthe activated saccharide ranges from 1 to 50, from 1 to 40, from 1 to30, from 1 to 20, from 1 to 10, from 1 to 5, from 3 to 40, from 3 to 30,from 3 to 20, from 3 to 10, from 4 to 40, from 4 to 30, from 4 to 20,from 4 to 10, from 5 to 30, from 5 to 25, from 5 to 20, from 5 to 10,from 6 to 50, from 6 to 40, from 6 to 30, from 6 to 20, from 6 to 15,from 6 to 14, from 6 to 13, from 6 to 12, from 6 to 11, from 6 to 10,from 7 to 40, from 7 to 30, from 7 to 20, from 7 to 15, from 7 to 14,from 7 to 13, from 7 to 12, from 7 to 11, from 7 to 10, from 8 to 40,from 8 to 30, from 8 to 20, from 8 to 15, from 8 to 14, from 8 to 13,from 8 to 13, from 8 to 12, from 8 to 11, from 8to 10, from 9to 40, from9to 30, from 9to 20, from 9to 15, from 10 to 40, from 10 to 30, from 10to 20, or from 10 to 15. In a further aspect, the degree of oxidation ofthe activated saccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40.

In a further aspect, the present disclosure provides a method of makinga glycoconjugate comprising a saccharide conjugated to a carrierprotein, comprising the steps of: a) reacting a saccharide with a stablenitroxyl or nitroxide radical compound, such as piperidine-N-oxy orpyrrolidine-N-oxy compounds, in the presence of an oxidant toselectively oxidize primary hydroxyls of the said saccharide to producean activated saccharide containing aldehyde groups; and b) reacting theactivated saccharide with a carrier protein comprising one or more aminegroups.

In said reaction, the actual oxidant is the N-oxoammonium salt, in acatalytic cycle. Preferably the stable nitroxyl or nitroxide radicalcompounds have the ability to selectively oxidize primary alcohol toaldehydes, in the presence of an oxidant, without over oxidation tocarboxylic acids.

In an aspect, step a) of the reaction is carried out in aqueous solvent.In another aspect, step a) is carried out in aprotic solvent. In anaspect, step a) is carried out in DMSO (dimethylsulfoxide),Dimethylacetamide (DMA), Sulfolane, N-Methyl-2-pyrrolidone (NMP),Hexamethylphosphoramide (HMPA) or in DMF (dimethylformamide) solvent.

In an aspect, the unreacted aldehyde groups are converted back toprimary alcohols during a capping step, using borohydride, afterconjugation with the carrier protein, therefore minimizing thesaccharide epitope modification during the modification steps involvingoxidation followed by conjugation.

In an aspect, said stable nitroxyl or nitroxide radical compound arepiperidine-N-oxy or pyrrolidine-N-oxy compounds. Preferably saidcompounds have the ability to selectively oxidize primary alcohols inthe presence of an oxidant, to generate aldehyde groups, withoutaffecting secondary hydroxyl groups. More preferably, said compoundshave the ability to selectively oxidize primary alcohol in the presenceof an oxidant, to generate aldehyde groups, without over oxidation tocarboxyl groups.

In an aspect, said stable nitroxyl or nitroxide radical compound bears aTEMPO or a PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety.Preferably said compound has the ability to selectively oxidize primaryalcohol in the presence of an oxidant, to generate aldehyde groups,without affecting secondary hydroxyl groups. More preferably, saidcompound has the ability to selectively oxidize primary alcohols in thepresence of an oxidant, to generate aldehyde groups, without overoxidation to carboxyl groups.

In an aspect, said stable nitroxyl radical compound is TEMPO or aderivative thereof. In an aspect, said stable nitroxyl radical compoundis selected from the group consisting of TEMPO,2,2,6,6-Tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy,4-Phosphonooxy-TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO,4-Isothiocyanato-TEMPO, 4-(2-Iodoacetamido)-TEMPO free radical,4-Hydroxy-TEMPO, 4-Cyano-TEMPO, 4-Carboxy-TEMPO,4-(2-Bromoacetamido)-TEMPO, 4-Amino-TEMPO,4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably said stablenitroxyl radical compound is TEMPO.

In a further aspect, said stable nitroxyl radical compound is selectedfrom the group consisting of 3β-DOXYL-5α-cholestane, 5-DOXYL-stearicacid, 16-DOXYL-stearic acid, Methyl 5-DOXYL-stearate,3-(Aminomethyl)-PROXYL, 3-Carbamoyl-PROXYL,3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, 3-Carboxy-PROXYL,3-Cyano-PROXYL.

In an aspect, said oxidant is a molecule bearing a N-halo moiety.Preferably said molecule has the ability to selectively oxidize primaryalcohol in the presence of a nitroxyl radical compound.

In an aspect, said oxidant is selected from the group consisting ofN-ChloroSuccinimide, N-Bromosuccinimide, N-Iodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

In an aspect, the degree of oxidation of the activated saccharide rangesfrom 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10,from 1 to 5, from 3 to 40, from 3 to 30, from 3 to 20, from 3 to 10,from 4 to 40, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30,from 5 to 25, from 5 to 20, from 5 to 10, from 6 to 50, from 6 to 40,from 6 to 30, from 6 to 20, from 6 to 15, from 6 to 14, from 6 to 13,from 6 to 12, from 6 to 11, from 6 to 10, from 7 to 40, from 7 to 30,from 7 to 20, from 7 to 15, from 7 to 14, from 7 to 13, from 7 to 12,from 7 to 11, from 7 to 10, from 8 to 40, from 8 to 30, from 8 to 20,from 8 to 15, from 8 to 14, from 8 to 13, from 8 to 13, from 8 to 12,from 8 to 11, from 8 to 10, from 9 to 40, from 9 to 30, from 9 to 20,from 9 to 15, from 10 to 40, from 10 to 30, from 10 to 20, or from 10 to15. In a further aspect, the degree of oxidation of the activatedsaccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or 40.

In an aspect, the saccharide is reacted with 0.1 to 10 molar equivalentsof oxidant. Preferably, the saccharide is reacted with 0.2 to 5, 0.5 to2.5 or 0.5 to 1.5 molar equivalent of oxidant. In an aspect, thepolysaccharide is reacted with about 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4,1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4,4.6, 4.8 or 5 molar equivalents of oxidant.

In an aspect, the stable nitroxyl or nitroxide radical compound ispresent in a catalytic amount. In an aspect, the saccharide is reactedwith less than about 0.3 molar equivalent of stable nitroxyl ornitroxide radical compound. In an aspect, the saccharide is reacted withless than about 0.005 molar equivalent of stable nitroxyl or nitroxideradical compound. In an aspect, the saccharide is reacted with about0.005, 0.01, 0.05 or 0.1 molar equivalent of stable nitroxyl ornitroxide radical compound.

In a further aspect, the saccharide is a bacterial capsularpolysaccharide. In another aspect the saccharide is a syntheticallyderived oligo or polysaccharide. In one aspect, the capsularpolysaccharide is derived from S. pneumonia (Pn). In a further aspect,the capsular polysaccharide is selected from Pn-serotype 10A,Pn-serotype 12F, and Pn-serotype 33F capsular polysaccharides. Forexample, in one aspect the capsular polysaccharide is a Pn-serotype 12Fcapsular polysaccharide.

In a further aspect, the capsular polysaccharide is derived from N.meningitidis. In one aspect, the capsular polysaccharide is selectedfrom meningococcal (Mn)-serotype A, C, W135, and Y capsularpolysaccharides.

In a further aspect, the capsular polysaccharide is meningococcal(Mn)-serotype X capsular polysaccharide.

In a further aspect, the capsular polysaccharide is derived from Group BStreptococcus (GBS). In one aspect, the capsular polysaccharide isselected from GBS serotypes Ia, Ib, II, III, IV, V, VI, VII and VIII.

In one aspect, the present disclosure provides any of the methodsdisclosed herein wherein the carrier protein is a toxin from tetanus,diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus orStreptococcus. In one aspect the carrier protein is CRM₁₉₇.

In a further aspect, the present disclosure provides a method asdescribed herein, wherein prior to step a), the saccharide is hydrolyzedto a molecular weight ranging from 100 to 400 kDa. For example, in oneaspect, the molecular weight ranges from 100 to 350 kDa, from 100 to 300kDa, from 100 to 250 kDa, from 100 to 200 kDa, from 100 to 150 kDa, from200 to 400 kDa, from 200 to 350 kDa, from 200 to 300 kDa, from 200 to250 kDa, from 300 to 400 kDa, or from 300 to 350 kDa.

In a further aspect, the present disclosure provides any of the methodsprovided herein further comprising the step of purifying the activatedpolysaccharide prior to step b). In a further aspect, the methodsfurther comprise the step of adding a reducing agent following step b).In one aspect, the reducing agent is NaCNBH₃. In a further aspect, themethods further comprise the step of adding NaBH₄ following the additionof NaCNBH₃. In a further aspect, the method comprises a purificationstep following the addition of NaBH₄.

In another aspect, the present disclosure provides a glycoconjugateproduced, or obtainable by any of the methods disclosed herein. Forexample, in one aspect the present disclosure provides a glycoconjugatecomprising a saccharide conjugated to a carrier protein that is producedor obtainable by the method comprising the steps of: a) reacting asaccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups. In a further aspect, thepresent disclosure provides a glycoconjugate comprising a saccharideconjugated to a carrier protein that is produced or obtainable by themethod comprising the steps of: a) reacting a saccharide with a stablenitroxyl or nitroxide radical compound and an oxidant to produce anactivated saccharide containing aldehyde groups; and b) reacting theactivated saccharide with a carrier protein comprising one or more aminegroups. Stable nitroxyl radical compounds and oxidant may be as definedat pages 2-4 above.

In a further aspect, the present disclosure provides an immunogeniccomposition comprising any of the glycoconjugates disclosed herein and apharmaceutically acceptable excipient, carrier, or diluent. In a furtheraspect, the immunogenic composition comprises an additional antigen. Ina further aspect, the additional antigen comprises a protein antigen ora glycoconjugate of a capsular polysaccharide derived from S.pneumoniae. For example, in one aspect the additional antigen comprisesa glycoconjugate of a capsular polysaccharide selected from Pn-serotypes1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F, 22F, and 23Fcapsular polysaccharides. In a further aspect, the additional antigencomprises a protein antigen or a glycoconjugate of a capsularpolysaccharide derived from N. meningitidis. In a further aspect, theadditional antigen comprises a glycoconjugate of a capsularpolysaccharide selected from serotypes A, C, W135 and Y capsularpolysaccharides. In a further aspect, the additional antigen comprises aglycoconjugate of a capsular polysaccharide from serotype X capsularpolysaccharides. In a further aspect, the additional antigen comprises aglycoconjugate of a capsular polysaccharide derived from Group BStreptococcus (GBS). In one aspect, the additional antigen comprises aglycoconjugate of a capsular polysaccharide selected from GBS serotypesIa, Ib, II, III, IV, V, VI, VII and VIII.

In a further aspect, the present disclosure provides any of theimmunogenic compositions disclosed herein, further comprising anadjuvant. In one aspect the adjuvant is an aluminum-based adjuvant. In afurther aspect, the aluminum-based adjuvant is selected from the groupconsisting of aluminum phosphate, aluminum sulfate, and aluminumhydroxide.

In another aspect, the present disclosure provides a method ofpreventing, treating or ameliorating a bacterial infection, disease orcondition in a subject, comprising administering to the subject animmunologically effective amount of any of the immunogenic compositionsdisclosed herein. In one aspect, the infection, disease or condition isassociated with S. pneumoniae bacteria. In a further aspect, theinfection, disease or condition is associated with N. meningitidisbacteria.

In another aspect, the present disclosure provides a method of inducinga protective immune response in a subject, comprising administering tothe subject an immunologically effective amount of any of theimmunogenic compositions disclosed herein.

In another aspect, the present disclosure provides an immunogeniccomposition comprising Pn-serotype 12F conjugated to a carrier proteinwherein the conjugate is stable. For example, in one aspect, the presentdisclosure provides an immunogenic composition comprising Pn-serotype12F conjugated to a carrier protein, wherein the amount of freePn-serotype 12F polysaccharide in the composition is less than 35% after120 days from when it was prepared. In a further aspect, the amount offree Pn-serotype 12F polysaccharide is less than 30%, less than 28%,less than 27%, less than 26%, or less than 25% after 120 days from whenit was prepared. In a further aspect, the amount of free Pn-serotype 12Fpolysaccharide is less than 35%, less than 30%, less than 28%, less than27%, less than 26%, or less than 25% after 90 days from when it wasprepared. In a further aspect, the amount of free Pn-serotype 12Fpolysaccharide is less than 35%, less than 30%, less than 28%, less than27%, less than 26%, or less than 25% after 60 days from when it wasprepared. In a further aspect, the amount of free Pn-serotype 12Fpolysaccharide is less than 35%, less than 30%, less than 28%, less than27%, less than 26%, or less than 25% after 30 days from when it wasprepared. In a further aspect, the present disclosure provides acomposition comprising Pn-serotype 3, 10A, or 33F conjugated to acarrier protein, wherein the amount of free Pn-serotype 3, 10A, or 33Fpolysaccharide, respectively, in the composition is less than 35% after120 days from when it was prepared. In a further aspect, the amount offree Pn-serotype 3, 10A, or 33F polysaccharide is less than 30%, lessthan 28%, less than 27%, less than 26%, or less than 25% after 120 daysfrom when it was prepared. In a further aspect, the amount of freePn-serotype 3, 10A, or 33F polysaccharide is less than 35%, less than30%, less than 28%, less than 27%, less than 26%, or less than 25% after90 days from when it was prepared. In a further aspect, the amount offree Pn-serotype 3, 10A, or 33F polysaccharide is less than 35%, lessthan 30%, less than 28%, less than 27%, less than 26%, or less than 25%after 60 days from when it was prepared. In a further aspect, the amountof free Pn-serotype 3, 10A, or 33F polysaccharide is less than 35%, lessthan 30%, less than 28%, less than 27%, less than 26%, or less than 25%after 30 days from when it was prepared. In one aspect, the amount offree polysaccharide as discussed above is measured at 25° C. In oneaspect, the carrier protein in the compositions disclosed above is atoxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli,Staphylococcus or Streptococcus. In a further aspect, the carrierprotein is CRM₁₉₇.

The present disclosure further provides an immunogenic compositioncomprising any of such glycoconjugates disclosed above and apharmaceutically acceptable excipient, carrier, or diluent. In a furtheraspect, such immunogenic compositions comprise an additional antigen.For example, in one aspect the additional antigen comprises a proteinantigen or a glycoconjugate of a capsular polysaccharide derived from S.pneumoniae. In a further aspect, the additional antigen comprises aglycoconjugate of a capsular polysaccharide selected from Pn-serotypes1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F, 22F, and 23Fcapsular polysaccharides. In an even further aspect, the additionalantigen comprises a protein antigen or a glycoconjugate of a capsularpolysaccharide derived from N. meningitidis. In an even further aspect,the additional antigen comprises a glycoconjugate of a capsularpolysaccharide selected from serotypes A, C, W135 and Y capsularpolysaccharides. In a further aspect, the additional antigen comprises aglycoconjugate of a capsular polysaccharide from serotype X capsularpolysaccharides. In a further aspect, the additional antigen comprises aglycoconjugate of a capsular polysaccharide from Group B Streptococcus(GBS). In one aspect, the capsular polysaccharide is selected from GBSserotypes Ia, Ib, II, III, IV, V, VI, VII and VIII.

In an even further aspect, such immunogenic compositions furthercomprise an adjuvant. For example, in one aspect the adjuvant is analuminum-based adjuvant. In a further aspect, the aluminum-basedadjuvant is selected from the group consisting of aluminum phosphate,aluminum sulfate, and aluminum hydroxide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the capsular polysaccharide of Pn-serotype12F.

FIG. 2 shows the dependence of N-Chlorosuccinimide in the Tempo/NCSoxidationation reaction on the degree of oxidation (DO).

FIG. 3 shows the structure of the capsular polysaccharide of Pn-serotype10A.

FIG. 4 shows the structure of the capsular polysaccharide of Pn-serotype33F.

FIG. 5 shows the structure of the capsular polysaccharide of Pn-serotype3.

FIG. 6 shows the putative mechanism of oxidation/conjugation ofPn-serotype 12F using TEMPO/NCS.

FIG. 7 shows the stability comparison of Pn-serotype 12F conjugatesprepared using periodate oxidation vs. TEMPO/NCS oxidation.

DETAILED DESCRIPTION

The present disclosure may be understood more readily by reference tothe following detailed description of the various embodiments of thedisclosure and the examples included herein. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thedisclosure pertains. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present disclosure, certain preferred methods andmaterials are described herein. In describing the embodiments and in theclaims, certain terminology will be used in accordance with thedefinitions set out below.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless indicated otherwise. Thus, for example, references to“the method” includes one or more methods, and/or steps of the typedescribed herein and/or which will become apparent to one of ordinaryskill in the art upon reading this disclosure.

As used herein, the term “about” means within a statistically meaningfulrange of a value, such as a stated concentration range, time frame,molecular weight, temperature or pH. Such a range can be within an orderof magnitude, typically within 20%, more typically within 10%, and evenmore typically within 5% or within 1% of a given value or range.Sometimes, such a range can be within the experimental error typical ofstandard methods used for the measurement and/or determination of agiven value or range. The allowable variation encompassed by the term“about” will depend upon the particular system under study, and can bereadily appreciated by one of ordinary skill in the art. Whenever arange is recited within this application, every whole number integerwithin the range is also contemplated as an embodiment of thedisclosure.

It is noted that in this disclosure, terms such as “comprises,”“comprised,” “comprising,” “contains,” “containing” and the like canhave the meaning attributed to them in U.S. patent law; e.g., they canmean “includes,” “included,” “including” and the like. Such terms referto the inclusion of particular ingredients or set of ingredients withoutexcluding any other ingredients. Terms such as “consisting essentiallyof” and “consists essentially of” have the meaning attributed to them inU.S. patent law, e.g., they allow for the inclusion of additionalingredients or steps that do not detract from the novel or basiccharacteristics of the disclosure, i.e., they exclude additionalunrecited ingredients or steps that detract from the novel or basiccharacteristics of the disclosure. The terms “consists of” and“consisting of” have the meaning ascribed to them in U.S. patent law;namely, that these terms are closed ended. Accordingly, these termsrefer to the inclusion of a particular ingredient or set of ingredientsand the exclusion of all other ingredients.

As used herein, the term “saccharide” may be used to refer to apolysaccharide, an oligosaccharide, or a monosaccharide.

As used herein, the term “degree of oxidation” in reference to asaccharide refers to the ratio of the moles of saccharide repeat unitper mole of aldehyde. The degree of oxidation of a saccharide can bedetermined using routine methods known to those of skill in the art.

The term “conjugates” or “glycoconjugates” as used herein refers to asaccharide covalently conjugated to a carrier protein. Glycoconjugatesof the disclosure and immunogenic compositions comprising them maycontain some amount of free saccharide.

The term “free saccharide” as used herein means a saccharide that is notcovalently conjugated to the carrier protein, but is neverthelesspresent in the glycoconjugate composition. The free saccharide may benon-covalently associated with (i.e., non-covalently bound to, adsorbedto, or entrapped in or with) the conjugated saccharide-carrier proteinglycoconjugate. The terms “free polysaccharide” and “free capsularpolysaccharide” may be used herein to convey the same meaning withrespect to glycoconjugates wherein the saccharide is a polysaccharide ora capsular polysaccharide, respectively.

As used herein, “to conjugate,” “conjugated” and “conjugating” refer toa process whereby a saccharide, for example a bacterial capsularpolysaccharide, is covalently attached to a carrier molecule or carrierprotein. The conjugation can be performed according to the methodsdescribed below or by other processes known in the art. Conjugationenhances the immunogenicity of the bacterial capsular polysaccharide.

The term “subject” refers to a mammal, including a human, or to a bird,fish, reptile, amphibian or any other animal. The term “subject” alsoincludes household pets or research animals. Non-limiting examples ofhousehold pets and research animals include: dogs, cats, pigs, rabbits,rats, mice, gerbils, hamsters, guinea pigs, ferrets, monkeys, birds,snakes, lizards, fish, turtles, and frogs. The term “subject” alsoincludes livestock animals. Non-limiting examples of livestock animalsinclude: alpaca, bison, camel, cattle, deer, pigs, horses, llamas,mules, donkeys, sheep, goats, rabbits, reindeer, yak, chickens, geese,and turkeys.

Glycoconjugates

The present disclosure relates to methods of preparing glycoconjugatescomprising a saccharide conjugated to a carrier protein, in particularby using a stable nitroxyl or nitroxide radial compound, to selectivelyoxidize primary alcohols of the saccharide to aldehydes, further usingan oxidant. In an aspect, said stable nitroxyl radical compound arepiperidine-N-oxy or pyrrolidine-N-oxy compounds. Preferably saidcompounds have the ability to selectively oxidize primary alcohol toaldehydes in the presence of an oxidant, without over oxidation tocarboxylic acids and without affecting secondary hydroxyl groups. In anaspect, said stable nitroxyl radical compound is a molecule bearing aTEMPO or a PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety.Preferably said molecule has the ability to selectively oxidize primaryalcohol in the presence of an oxidant, to generate aldehyde groups,without affecting secondary hydroxyl groups. More preferably saidmolecule has the ability to selectively oxidize primary alcohol in thepresence of an oxidant, to generate aldehyde groups, without overoxidation to carboxyl groups. In an aspect, said stable nitroxyl radicalcompound is selected from the groups consisting of TEMPO,2,2,6,6-Tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy,4-Phosphonooxy-TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO,4-Isothiocyanato-TEMPO, 4-(2-Iodoacetamido)-TEMPO free radical,4-Hydroxy-TEMPO, 4-Cyano-TEMPO, 4-Carboxy-TEMPO,4-(2-Bromoacetamido)-TEMPO, 4-Amino-TEMPO,4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably said stablenitroxyl radical compound is TEMPO. In a further aspect, said stablenitroxyl radical compound is selected from the groups consisting of3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid,Methyl 5-DOXYL-stearate, 3-(Aminomethyl)-PROXYL, 3-Carbamoyl-PROXYL,3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, 3-Carboxy-PROXYL,3-Cyano-PROXYL. In an aspect, the oxidant is a molecule bearing a N-halomoiety. Preferably said molecule has the ability to selectively oxidizeprimary alcohol in the presence of a nitroxyl radical compound. In anaspect, said oxidant is selected from the group consisting ofN-Chlorosuccinimide, N-Bromosuccinimide, N-Iodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

In an aspect, the present disclosure relates to methods of preparingglycoconjugates comprising a saccharide conjugated to a carrier protein,in particular by using 2,2,6,6-Tetramethyl-1-piperidinyloxy free radical(TEMPO) to oxidize primary alcohols of the saccharide to aldehydes usingN-Chlorosuccinimide (NCS) as the cooxidant.

In the glycoconjugates of the disclosure, the saccharide is selectedfrom the group consisting of a polysaccharide, an oligosaccharide, and amonosaccharide, and the carrier protein is selected from any suitablecarrier as further described herein or known to those of skill in theart. In some embodiments, the saccharide is a polysaccharide, inparticular a bacterial capsular polysaccharide, such as Streptococcuspneumoniae serotype 10A (Pn-serotype 10A), Pn-serotype 12F, orPn-serotype 33F. In some such embodiments, the carrier protein isCRM₁₉₇.

Capsular polysaccharides can be prepared by standard techniques known tothose skilled in the art. For example, capsular polysaccharides can beprepared from a variety of serotypes, such as 1, 3, 4, 5, 6A, 6B, 7F, 8,9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F ofStreptococcus pneumoniae. These pneumococcal conjugates are prepared byseparate processes and formulated into a single dosage formulation. Forexample, in one embodiment, each pneumococcal polysaccharide serotype isgrown in a soy-based medium. The individual polysaccharides are thenpurified through centrifugation, precipitation, ultra-filtration, andcolumn chromatography. The purified polysaccharides are chemicallyactivated to make the saccharides (i.e. activated saccharides) capableof reacting with the carrier protein. Once activated, each capsularpolysaccharide is separately conjugated to a carrier protein to form aglycoconjugate. In one embodiment, each capsular polysaccharide isconjugated to the same carrier protein. The chemical activation of thepolysaccharides and subsequent conjugation to the carrier protein can beachieved by conventional means. See, for example, U.S. Pat. Nos.4,673,574, 4,902,506, 7,709,001, and 7,955,605.

In one embodiment, the glycoconjugate of the disclosure has a molecularweight of between about 50 kDa and about 20,000 kDa. In anotherembodiment, the glycoconjugate has a molecular weight of between about200 kDa and about 10,000 kDa. In another embodiment, the glycoconjugatehas a molecular weight of between about 500 kDa and about 5,000 kDa. Inone embodiment, the glycoconjugate has a molecular weight of betweenabout 1,000 kDa and about 3,000 kDa. In other embodiments theglycoconjugate has a molecular weight of between about 600 kDa and about2800 kDa; between about 700 kDa and about 2700 kDa; between about 1000kDa and about 2000 kDa; between about 1800 kDa and about 2500 kDa;between about 1100 kDa and about 2200 kDa; between about 1900 kDa andabout 2700 kDa; between about 1200 kDa and about 2400 kDa; between about1700 kDa and about 2600 kDa; between about 1300 kDa and about 2600 kDa;between about 1600 kDa and about 3000 kDa. Any whole number integerwithin any of the above ranges is contemplated as an embodiment of thedisclosure.

Novel features of the glycoconjugates of the disclosure include themolecular weight profiles of the saccharides and resulting conjugates,the ratio of conjugated lysines per carrier protein and number oflysines covalently linked to the polysaccharide, the number of covalentlinkages between the carrier protein and the saccharide as a function ofrepeat units of the saccharide, and the relative amount of freesaccharide compared to the total saccharide.

In another embodiment, the polysaccharide is a capsular polysaccharidederived from Neisseria meningitidis. In some such embodiments, thecapsular polysaccharide is selected from the group consisting ofserotype A, B, C, W135, X and Y capsular polysaccharides of N.meningitidis. In one such embodiment, the capsular polysaccharide is aserotype C capsular polysaccharide. In another such embodiment, thecapsular polysaccharide is a serotype W135 capsular polysaccharide. Inanother such embodiment, the capsular polysaccharide is a serotype Ycapsular polysaccharide.

In some embodiments, the glycoconjugate of the disclosure comprises abacterial capsular polysaccharide, wherein the capsular polysaccharidehas a molecular weight of between 10 kDa and 2,000 kDa or between 50 kDaand 1,000 kDa. In some such embodiments, the capsular polysaccharide isderived from S. pneumoniae or N. meningitidis. In some such embodiments,the capsular polysaccharide is derived from S. pneumoniae and isselected from the group consisting of serotype 1, 3, 4, 5, 6A, 6B, 7F,8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsularpolysaccharides. In other such embodiments, the capsular polysaccharideis derived from N. meningitidis and is selected from the groupconsisting of serotype A, B, C, W135, X and Y capsular polysaccharides.

In one embodiment, the disclosure provides a glycoconjugate comprising acapsular polysaccharide covalently conjugated to a carrier protein,having one or more of the following features: the polysaccharide has amolecular weight of between 50 kDa and 1,000 kDa; the glycoconjugate hasa molecular weight of between 1,000 kDa to 3,000 KDa; and the conjugatecomprises less than about 45% free polysaccharide relative to totalpolysaccharide. In some embodiments, the polysaccharide has a molecularweight of between 10 kDa and 2,000 kDa. In some embodiments theglycoconjugate has a molecular weight of between 50 kDa and 20,000 kDa.In other embodiments the glycoconjugate has a molecular weight ofbetween 200 kDa and 10,000 kDa. In other embodiments, the conjugatecomprises less than about 30%, 20%, 15%, 10%, or 5% free polysacchariderelative to total polysaccharide. The amount of free polysaccharide canbe measured as a function of time, for example after 10, 20, 30, 40, 50,60, 70, 80, 90, or 120 days, or even longer, after the conjugate wasprepared.

The number of lysine residues in the carrier protein conjugated to thesaccharide can be characterized as a range of conjugated lysines, whichmay be expressed as a molar ratio. For example, in an immunogeniccomposition where 4 to 15 lysine residues of CRM₁₉₇ are covalentlylinked to the saccharide, the molar ratio of conjugated lysines toCRM₁₉₇ in the glycoconjugate is between about 10:1 to about 40:1. In animmunogenic composition where 2 to 20 lysine residues of CRM₁₉₇ arecovalently linked to the saccharide, the molar ratio of conjugatedlysines to CRM₁₉₇ in the glycoconjugate is between about 5:1 to about50:1.

In one embodiment, the molar ratio of conjugated lysines to carrierprotein is from about 10:1 to about 25:1. In some such embodiments, thecarrier protein is CRM₁₉₇. In one embodiment, the saccharide:carrierprotein ratio (w/w) is between 0.2 and 4. In another embodiment, thesaccharide:carrier protein ratio (w/w) is between 1.1 and 1.7. In someembodiments, the saccharide is a bacterial capsular polysaccharide, andthe saccharide:carrier protein ratio (w/w) is between 0.2 and 4. Inother embodiments, the saccharide is a bacterial capsularpolysaccharide, and the saccharide:carrier protein ratio (w/w) isbetween 1.1 and 1.7. In some such embodiments, the carrier protein isCRM₁₉₇.

The frequency of attachment of the saccharide chain to a lysine on thecarrier protein is another parameter for characterizing theglycoconjugates of the disclosure. For example, in one embodiment, thereis at least one covalent linkage between the carrier protein and thepolysaccharide for every 100 saccharide repeat units of thepolysaccharide. In one embodiment, there is at least one covalentlinkage between the carrier protein and the polysaccharide for every 50saccharide repeat units of the polysaccharide. In one embodiment, thereis at least one covalent linkage between the carrier protein and thepolysaccharide for every 25 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 4saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 10 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every15 saccharide repeat units of the polysaccharide.

In frequent embodiments, the carrier protein is CRM₁₉₇ and the covalentlinkage between the CRM₁₉₇ and the polysaccharide occurs at least oncein every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide.In some such embodiments, the polysaccharide is a bacterial capsularpolysaccharide, for example a capsular polysaccharide derived from S.pneumoniae or N. meningitidis bacteria.

In other embodiments, the conjugate comprises at least one covalentlinkage between the carrier protein and saccharide for every 5 to 10saccharide repeat units; every 2 to 7 saccharide repeat units; every 3to 8 saccharide repeat units; every 4 to 9 saccharide repeat units;every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeatunits; every 8 to 13 saccharide repeat units; every 9 to 14 sacchariderepeat units; every 10 to 15 saccharide repeat units; every 2 to 6saccharide repeat units, every 3 to 7 saccharide repeat units; every 4to 8 saccharide repeat units; every 6 to 10 saccharide repeat units;every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeatunits; every 9 to 13 saccharide repeat units; every 10 to 14 sacchariderepeat units; every 10 to 20 saccharide repeat units; or every 4 to 25saccharide repeat units.

In another embodiment, at least one linkage between carrier protein andsaccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units ofthe polysaccharide.

In one embodiment, the glycoconjugate of the disclosure comprises atleast one covalent linkage between the carrier protein and thepolysaccharide for every 25 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 4saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 10 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every15 saccharide repeat units of the polysaccharide.

In one embodiment, the glycoconjugate comprises less than about 45% freesaccharide compared to the total amount of saccharide. In anotherembodiment, the glycoconjugate comprises less than about 30% freesaccharide compared to the total amount of saccharide. In anotherembodiment, the glycoconjugate comprises less than about 20% freesaccharide compared to the total amount of saccharide. In a furtherembodiment, the glycoconjugate comprises less than about 10% freesaccharide compared to the total amount of saccharide. In anotherembodiment, the glycoconjugate comprises less than about 5% freesaccharide compared to the total amount of saccharide.

In another embodiment, the glycoconjugate comprises less than about 20mole % of carrier protein residues compared to the total amount ofglycoconjugate.

In another aspect, the disclosure provides an immunogenic compositioncomprising a glycoconjugate of the disclosure and at least one of anadjuvant, diluent or carrier.

In one embodiment, the disclosure provides an immunogenic compositioncomprising a glycoconjugate of the disclosure and at least one of anadjuvant, diluent or carrier, wherein the glycoconjugate comprises abacterial capsular polysaccharide covalently conjugated to a carrierprotein. In some such embodiments, the capsular polysaccharide isderived from S. pneumoniae or N. meningitidis.

In some embodiments, the immunogenic composition comprises an adjuvant.In some such embodiments, the adjuvant is an aluminum-based adjuvantselected from the group consisting of aluminum phosphate, aluminumsulfate and aluminum hydroxide. In one embodiment, the immunogeniccomposition comprises the adjuvant aluminum phosphate.

In some embodiments, the glycoconjugates or immunogenic compositions ofthe disclosure can be used to generate antibodies that are functional asmeasured by killing bacteria in an animal efficacy model or via anopsonophagocytic killing assay.

In one embodiment, the disclosure provides a method of inducing animmune response in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic composition of thedisclosure as described herein. In another aspect, the disclosureprovides a method for inducing an immune response against a pathogenicbacterium in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic composition asdescribed herein. In another aspect, the disclosure provides a methodfor preventing or ameliorating a disease or condition caused by apathogenic bacterium in a subject, comprising administering to thesubject an immunologically effective amount of an immunogeniccomposition as described herein. In another aspect, the disclosureprovides a method for reducing the severity of at least one symptom of adisease or condition caused by infection with a pathogenic bacterium ina subject, comprising administering to the subject an immunologicallyeffective amount of an immunogenic composition as described herein. Insome embodiments, the pathogenic bacterium is S. pneumoniae or N.meningitidis.

In addition, the present disclosure provides methods for inducing animmune response against S. pneumoniae or N. meningitidis bacteria,methods for preventing a disease caused by S. pneumoniae or N.meningitidis bacteria, and methods for reducing the severity of at leastone symptom of a disease caused by infection with S. pneumoniae or N.meningitidis bacteria.

Saccharides

Saccharides include polysaccharides, oligosaccharides andmonosaccharides. In some embodiments, the saccharide is apolysaccharide, in particular a bacterial capsular polysaccharide.

The molecular weight of the capsular polysaccharide is a considerationfor use in immunogenic compositions. High molecular weight capsularpolysaccharides are able to induce certain antibody immune responses dueto a higher valence of the epitopes present on the antigenic surface.The isolation and purification of high molecular weight capsularpolysaccharides is contemplated for use in the conjugates, compositionsand methods of the present disclosure.

In one embodiment, the capsular polysaccharide has a molecular weight ofbetween 10 kDa and 2,000 kDa. In one embodiment, the capsularpolysaccharide has a molecular weight of between 50 kDa and 1,000 kDa.In another embodiment, the capsular polysaccharide has a molecularweight of between 50 kDa to 300 kDa. In another embodiment, the capsularpolysaccharide has a molecular weight of between 70 kDa to 300 kDa. Infurther embodiments, the capsular polysaccharide has a molecular weightof between 90 kDa to 250 kDa; 90 kDa to 150 kDa; 90 kDa to 120 kDa; 80kDa to 120 kDa; 70 kDa to 100 kDa; 70 kDa to 110 kDa; 70 kDa to 120 kDa;70 kDa to 130 kDa; 70 kDa to 140 kDa; 70 kDa to 150 kDa; 70 kDa to 160kDa; 80 kDa to 110 kDa; 80 kDa to 120 kDa; 80 kDa to 130 kDa; 80 kDa to140 kDa; 80 kDa to 150 kDa; 80 kDa to 160 kDa; 90 kDa to 110 kDa; 90 kDato 120 kDa; 90 kDa to 130 kDa; 90 kDa to 140 kDa; 90 kDa to 150 kDa; 90kDa to 160 kDa; 100 kDa to 120 kDa; 100 kDa to 130 kDa; 100 kDa to 140kDa; 100 kDa to 150 kDa; 100 kDa to 160 kDa; and similar desiredmolecular weight ranges. Any whole number integer within any of theabove ranges is contemplated as an embodiment of the disclosure.

The capsular polysaccharide of S. pneumoniae, Serotype 12F (Pn-serotype12F) has the structure shown in FIG. 1. The capsular polysaccharide ofS. pneumoniae, Serotype 10A (Pn-serotype 10A) has the structure shown inFIG. 3. The capsular polysaccharide of S. pneumoniae, Serotype 33F(Pn-serotype 33F) has the structure shown in FIG. 4. The capsularpolysaccharide of S. pneumoniae, Serotype 3 (Pn-serotype 3) has thestructure shown in FIG. 5.

In some embodiments, the capsular polysaccharides, glycoconjugates orimmunogenic compositions of the disclosure are used to generateantibodies that are functional as measured by the killing of bacteria inan animal efficacy model or an opsonophagocytic killing assay thatdemonstrates that the antibodies kill the bacteria. Capsularpolysaccharides can be obtained directly from bacteria using isolationprocedures known to one of ordinary skill in the art. See, e.g.,Fournier et al. (1984), supra; Fournier et al. (1987) Ann. Inst.Pasteur/Microbiol. 138:561-567; US Patent Application Publication No,2007/0141077; and Intl Patent Application Publication No. WO 00/56357;each of which is incorporated herein by reference as if set forth in itsentirety). In addition, they can be produced using synthetic protocols.Moreover, capsular polysaccharide can be recombinantly produced usinggenetic engineering procedures also known to one of ordinary skill inthe art (see, Sau et al. (1997) Microbiology 143:2395-2405; and U.S.Pat. No. 6,027,925; each of which is incorporated herein by reference asif set forth in its entirety). S. pneumoniae or N. menigitidis strainscan be used to make the respective polysaccharides that are obtainedeither from established culture collections or clinical specimens.

Carrier Proteins

Another component of the glycoconjugate of the disclosure is a carrierprotein to which the saccharide is conjugated. The term “proteincarrier” or “carrier protein” or “carrier” refers to any proteinmolecule that may be conjugated to an antigen (such as a capsularpolysaccharide) against which an immune response is desired.

Conjugation to a carrier can enhance the immunogenicity of the antigen.Protein carriers for the antigens can be toxins, toxoids or any mutantcross-reactive material (CRM) of the toxin from tetanus, diphtheria,pertussis, Pseudomonas, E. coli, Staphylococcus and Streptococcus. Inone embodiment, a carrier is of diphtheria toxoid CRM₁₉₇, derived fromC. diphtheriae strain C7 (6197), which produces CRM₁₉₇ protein. Thisstrain has ATCC accession No. 53281. A method for producing CRM₁₉₇ isdescribed in U.S. Pat. No. 5,614,382, which is incorporated herein byreference as if set forth in its entirety. Alternatively, a fragment orepitope of the protein carrier or other immunogenic protein can be used.For example, a haptenic antigen can be coupled to a T-cell epitope of abacterial toxin, toxoid or CRM. Other suitable carrier proteins includeinactivated bacterial toxins such as cholera toxoid (e.g., as describedin Intl Patent Application No. WO 2004/083251), E. coli LT, E. coli ST,and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membraneproteins such as outer membrane complex c (OMPC), porins, transferrinbinding proteins, pneumolysin, pneumococcal surface protein A (PspA),pneumococcal adhesion protein (PsaA) or Haemophilus influenzae protein Dcan also be used. Other proteins, such as ovalbumin, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD) also can be used as carrier proteins.

As discussed previously herein, the number of lysine residues in thecarrier protein that become conjugated to the saccharide can becharacterized as a range of conjugated lysines. For example, in a givenimmunogenic composition, the CRM₁₉₇ may comprise 1 to 15 lysine residuesout of 39 covalently linked to the saccharide. Another way to expressthis parameter is that about 2.5% to about 40% of CRM₁₉₇ lysines arecovalently linked to the saccharide. For example, in a given immunogeniccomposition, the CRM₁₉₇ may comprise 1 to 20 lysine residues out of 39covalently linked to the saccharide. Another way to express thisparameter is that about 2.5% to about 50% of CRM₁₉₇ lysines arecovalently linked to the saccharide.

Methods for Making Glycoconjugates

In order to make a glycoconjugate, a polysaccharide must first beactivated (i.e. chemically modified) before it can be chemically linkedto a carrier, such as a protein. Prior to the activation step,saccharides can be hydrolyzed or mechanically sized by pressurehomogenization to achieve appropriate molecular weights (e.g. 50 kDa to500 kDa) for activation and subsequent conjugation. Partial oxidation ofcarbohydrates in polysaccharides has been effectively utilized togenerate aldehyde groups which are then coupled to amine groups, such asthe lysine residues of carrier proteins, to generate immunogenicconjugates. It is important that the method used to conjugate apolysaccharide to a carrier protein results in a stable covalentlinkage, and the reaction conditions are mild enough to maintain thestructural integrity of the individual components. Methods commonly usedto activate and couple polysaccharides to carrier proteins includereductive amination chemistry (RAC), cyanylation, and use ofcarbodiimide. Reductive amination typically involves the use of sodiumor potassium periodate or periodic acid in order to selectively oxidizevicinal —OH groups into active aldehyde groups. Cyanylation is used torandomly convert —OH groups into active —CN groups. Carbodiimide is usedto activate carboxyl groups by replacing —OH groups with carbodiimide.

Reductive amination chemistry (RAC) is one of the most common methodsused to couple polysaccharides to proteins since the reaction betweenthe resulting carbonyl group of the polysaccharide and the amino groupof the carrier protein can form corresponding Schiffs' base, which canthen be selectively reduced in the presence of sodium cyanoborohydride(NaCNBH₃) to a very stable saturated carbon-nitrogen bond. Furthermore,reductive amination can be carried out in aqueous solution underconditions mild enough to preserve the structural integrity of thesaccharide and protein components. Following conjugation, unreactedaldehydes can then be capped via sodium borohydride (NaBH₄) reduction.The conjugate can then be purified (e.g., byultrafiltration/diafiltration), giving a final bulk glycoconjugate insuccinate buffered saline.

However, depending on the particular polysaccharide, use of the commonmethods noted above does not always provide adequate results. Forexample, direct oxidation of polysaccharides with sodium periodate canresult in result in the cleavage of the polysaccharide backbone.

For example, it was observed that for the conjugates prepared usingstandard periodate oxidation conditions (followed by reductiveamination), representative batches showed an increase in freepolysaccharide and a reduction in molecular weight, at 25° C. and above.The present disclosure provides the finding that the use of anN-oxoammonium salt based oxidation method resulted in improved stabilityof several S. pneumoniae polysaccharide conjugates, particularlySerotype 12F. In particular, as shown in further detail in Examples 1 to7, the free radical 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) wasused in combination with N-chlorosuccinimide (NCS) to effectivelyoxidize the primary hydroxyl groups of Serotypes 12F, 10A, 3 and 33F inorder to improve the stability of the resulting conjugates. Althoughselective oxidation of primary alcohols to aldehydes using TEMPO/NCS hasbeen shown in the context of organic chemical reactions using smallmolecules in organic solvents (see, e.g. Einhorn et al., J. Org. Chem.61, pp. 7452-7454 (1996)), the present disclosure provides the novelfinding that TEMPO/NCS can be used as an oxidizing agent to selectivelyoxidize complex polysaccharides in aqueous solution in order to producestable polysaccharide protein conjugates.

Accordingly, in one embodiment, the present disclosure provides methodsof making glycoconjugates that comprise a saccharide conjugated to acarrier protein, comprising the steps of: a) reacting a saccharide witha stable nitroxyl radical compound and an oxidant to produce anactivated saccharide; and b) reacting the activated saccharide with acarrier protein comprising one or more amine groups.

In an aspect, the unreacted aldehyde groups are converted back toprimary alcohols during a capping step, using borohydride, afterconjugation with the carrier protein, therefore minimizing thesaccharide epitope modification during the modification steps involvingoxidation followed by conjugation.

In an aspect, step a) of the reaction is carried out in aqueous solvent.In another aspect, step a) is carried out in aprotic solvent. In anaspect, step a) is carried out in DMSO (dimethylsulfoxide),Dimethylacetamide (DMA), Sulfolane, N-Methyl-2-pyrrolidone (NMP),Hexamethylphosphoramide (HMPA) or in DMF (dimethylformamide) solvent.

In an aspect, said stable nitroxyl radical compound are piperidine-N-oxyor pyrrolidine-N-oxy compounds. Preferably said compounds have theability to selectively oxidize primary alcohols in the presence of anoxidant, to generate aldehyde groups, without affecting secondaryhydroxyl groups. More preferably, said compounds have the ability toselectively oxidize primary alcohol in the presence of an oxidant, togenerate aldehyde groups, without over oxidation to carboxyl groups. Inan embodiment, said stable nitroxyl radical compound is a moleculebearing a TEMPO or a PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy)moiety. Preferably said molecule has the ability to selectively oxidizeprimary alcohol in the presence of an oxidant, to generate aldehydegroups, without affecting secondary hydroxyl groups. More preferablysaid molecule has the ability to selectively oxidize primary alcohols inthe presence of an oxidant, to generate aldehyde groups, without overoxidation to carboxyl groups. In an aspect, said stable nitroxyl radicalcompound is TEMPO or a derivative thereof. In an embodiment, said stablenitroxyl radical compound is selected from the groups consisting ofTEMPO, 2,2,6,6-Tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy,4-Phosphonooxy-TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO,4-Isothiocyanato-TEMPO, 4-(2-Iodoacetamido)-TEMPO free radical,4-Hydroxy-TEMPO, 4-Cyano-TEMPO, 4-Carboxy-TEMPO,4-(2-Bromoacetamido)-TEMPO, 4-Amino-TEMPO,4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably said stablenitroxyl radical compound is TEMPO. In a further embodiment, said stablenitroxyl radical compound is selected from the groups consisting of3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid,Methyl 5-DOXYL-stearate, 3-(Aminomethyl)-PROXYL, 3-Carbamoyl-PROXYL,3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, 3-Carboxy-PROXYL,3-Cyano-PROXYL. In an embodiment, said oxidant is a molecule bearing aN-halo moiety. Preferably said molecule has the ability to selectivelyoxidize primary alcohol in the presence of a nitroxyl radical compound.In an embodiment, said oxidant is selected from the group consisting ofN-Chlorosuccinimide, N-Bromosuccinimide, N-Iodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

In an aspect, the saccharide is reacted with 0.1 to 10 molar equivalentof oxidant. Preferably, the saccharide is reacted with 0.2 to 5, 0.5 to2.5 or 0.5 to 1.5 molar equivalent of oxidant. In an aspect, thepolysaccharide is reacted with about 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4,1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4,4.6, 4.8 or 5 molar equivalent of oxidant.

In an aspect, the stable nitroxyl radical compound is present in acatalytic amount. In an aspect, the saccharide is reacted with less thanabout 0.3 molar equivalent of stable nitroxyl radical compound. In anaspect, the saccharide is reacted with less than about 0.005 molarequivalent of stable nitroxyl radical compound. In an aspect, thesaccharide is reacted with about 0.005, 0.01, 0.05 or 0.1 molarequivalent of stable nitroxyl radical compound.

In one embodiment, the present disclosure provides methods of makingglycoconjugates that comprise a saccharide conjugated to a carrierprotein, comprising the steps of: a) reacting a saccharide with2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide(NCS) in an aqueous solvent to produce an activated saccharide; and b)reacting the activated saccharide with a carrier protein comprising oneor more amine groups.

In other embodiments, the method further comprises a step of purifyingthe glycoconjugate, for example, by diafiltration.

In each case, the saccharide is selected from the group consisting of apolysaccharide, an oligosaccharide and a monosaccharide.

In each case, the said saccharide may be purified from the fermentationmedium or synthetically derived.

In frequent embodiments, the carrier protein is CRM₁₉₇. In oneembodiment, the bacterial capsular polysaccharide is a capsularpolysaccharide derived from S. pneumoniae. In another preferredembodiment, the bacterial capsular polysaccharide is a capsularpolysaccharide derived from N. meningitides.

In one embodiment the method of producing a glycoconjugate of thedisclosure comprises the step of isolating the saccharide-carrierprotein conjugate after it is produced. In frequent embodiments, theglycoconjugate is isolated by ultrafiltration.

In one embodiment, the carrier protein used in the method of producingan isolated S. pneumoniae capsular polysaccharide-carrier proteinconjugate comprises CRM₁₉₇. In one embodiment, the carrier protein usedin the method of producing an isolated N. meningitidis capsularpolysaccharide-carrier protein conjugate comprises CRM₁₉₇.

In one embodiment, the CRM₁₉₇ is reacted with the activatedpolysaccharide at a ratio by weight of about 1:1.

In one embodiment, the method of producing an isolated S. pneumoniaecapsular polysaccharide:carrier protein conjugate comprises the step ofcapping the polysaccharide-carrier protein conjugate reaction mixture toremove unreacted activation groups.

In one embodiment, the CRM₁₉₇ in the method of producing capsularpolysaccharide-CRM₁₉₇ conjugate is added in a ratio by weight of about0.4:1 CRM₁₉₇:capsular polysaccharide molecule. In other embodiments, theratio by weight of CRM₁₉₇:capsular polysaccharide is about 0.5:1, about0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1.1:1,about 1.2:1, about 1.3:1, about 1.4:1, or about 1.5:1.

In one embodiment, the saccharide used in the method of producing theglycoconjugate of the disclosure has a molecular weight between about 10kDa and about 2,000 kDa. In other embodiments, the molecular weight isbetween about 50 kDa and about 1,000 kDa, between about 50 kDa and about20,000 kDa, between about 200 kDa and about 10,000 kDa, between about1,000 kDa and about 3,000 kDa.

In another aspect, the disclosure provides an immunogenic compositioncomprising a glycoconjugate produced by any of the methods describedherein.

In another aspect, the disclosure provides an immunogenic compositioncomprising a glycoconjugate obtainable by any of the methods describedherein.

Immunogenic Compositions

The term “immunogenic composition” relates to any pharmaceuticalcomposition containing an antigen, e.g., a microorganism or a componentthereof, which composition can be used to elicit an immune response in asubject.

As used herein, “immunogenic” means an ability of an antigen (or anepitope of the antigen), such as a bacterial capsular polysaccharide, ora glycoconjugate or immunogenic composition comprising the antigen, toelicit an immune response in a host such as a mammal, either humorallyor cellularly mediated, or both.

Accordingly, a “glycoconjugate” or “conjugate” as used herein means anyglycoconjugate containing an antigen or antigenic determinant (i.e.,epitope) of a bacterial capsular polysaccharide conjugated to a carriermolecule that can be used to elicit an immune response.

The glycoconjugate may serve to sensitize the host by the presentationof the antigen in association with MHC molecules at a cell surface. Inaddition, antigen-specific T-cells or antibodies can be generated toallow for the future protection of an immunized host. Glycoconjugatesthus can protect the host from one or more symptoms associated withinfection by the bacteria, or may protect the host from death due to theinfection with the bacteria associated with the capsular polysaccharide.Glycoconjugates may also be used to generate polyclonal or monoclonalantibodies, which may be used to confer passive immunity to a subject.Glycoconjugates may also be used to generate antibodies that arefunctional as measured by the killing of bacteria in either an animalefficacy model or via an opsonophagocytic killing assay.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, unlessotherwise indicated by context, the term is intended to encompass notonly intact polyclonal or monoclonal antibodies, but also engineeredantibodies (e.g., chimeric, humanized and/or derivatized to altereffector functions, stability and other biological activities) andfragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv)and domain antibodies, including shark and camelid antibodies), andfusion proteins comprising an antibody portion, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies so long as theyexhibit the desired biological activity) and antibody fragments asdescribed herein, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site. Anantibody includes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 inhumans. The heavy-chain constant domains that correspond to thedifferent classes of immunoglobulins are called alpha, delta, epsilon,gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody.

The term “antigen” generally refers to a biological molecule, usually aprotein, peptide, polysaccharide or conjugate in an immunogeniccomposition, or immunogenic substance that can stimulate the productionof antibodies or T-cell responses, or both, in an animal, includingcompositions that are injected or absorbed into an animal. The immuneresponse may be generated to the whole molecule, or to a variousportions of the molecule (e.g., an epitope or hapten). The term may beused to refer to an individual molecule or to a homogeneous orheterogeneous population of antigenic molecules. An antigen isrecognized by antibodies, T-cell receptors or other elements of specifichumoral and/or cellular immunity. “Antigen” also includes all relatedantigenic epitopes. Epitopes of a given antigen can be identified usingany number of epitope mapping techniques, well known in the art. See,e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66(Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example,linear epitopes may be determined by, e.g., concurrently synthesizinglarge numbers of peptides on solid supports, the peptides correspondingto portions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;Geysen et al. (1986) Molec. Immunol. 23:709-715; each of which isincorporated herein by reference as if set forth in its entirety.Similarly, conformational epitopes may be identified by determiningspatial conformation of amino acids such as by, e.g., x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, supra.

Furthermore, for purposes of the present disclosure, “antigen” also canbe used to refer to a protein that includes modifications, such asdeletions, additions and substitutions (generally conservative innature, but they may be non-conservative), to the native sequence, aslong as the protein maintains the ability to elicit an immunologicalresponse. These modifications may be deliberate, as throughsite-directed mutagenesis, or through particular synthetic procedures,or through a genetic engineering approach, or may be accidental, such asthrough mutations of hosts, which produce the antigens. Furthermore, theantigen can be derived, obtained, or isolated from a microbe, e.g., abacterium, or can be a whole organism. Similarly, an oligonucleotide orpolynucleotide, which expresses an antigen, such as in nucleic acidimmunization applications, is also included in the definition. Syntheticantigens are also included, e.g., polyepitopes, flanking epitopes, andother recombinant or synthetically derived antigens (Bergmann et al.(1993) Eur. J. Immunol. 23:2777 2781; Bergmann et al. (1996) J. Immunol.157:3242-3249; Suhrbier (1997) Immunol. Cell Biol. 75:402 408; Gardneret al. (1998) 12th World AIDS Conference, Geneva, Switzerland, Jun. 28to Jul. 3, 1998).

A “protective” immune response refers to the ability of an immunogeniccomposition to elicit an immune response, either humoral or cellmediated, or both, which serves to protect a subject from an infection.The protection provided need not be absolute, i.e., the infection neednot be totally prevented or eradicated, if there is a statisticallysignificant improvement compared with a control population of subjects,e.g. infected animals not administered the vaccine or immunogeniccomposition. Protection may be limited to mitigating the severity orrapidity of onset of symptoms of the infection. In general, a“protective immune response” would include the induction of an increasein antibody levels specific for a particular antigen in at least 50% ofsubjects, including some level of measurable functional antibodyresponses to each antigen. In particular situations, a “protectiveimmune response” could include the induction of a two fold increase inantibody levels or a fourfold increase in antibody levels specific for aparticular antigen in at least 50% of subjects, including some level ofmeasurable functional antibody responses to each antigen. In certainembodiments, opsonising antibodies correlate with a protective immuneresponse. Thus, protective immune response may be assayed by measuringthe percent decrease in the bacterial count in an opsonophagocytosisassay, for instance those described below. Preferably, there is adecrease in bacterial count of at least 10%, 25%, 50%, 65%, 75%, 80%,85%, 90%, 95% or more. The “immunogenic amount” of a particularconjugate in a composition is generally dosed based on totalpolysaccharide, conjugated and non-conjugated for that conjugate. Forexample, a capsular polysaccharide conjugate with 20% freepolysaccharide will have about 80 mcg of conjugated polysaccharide andabout 20 mcg of non-conjugated polysaccharide in a 100 mcg dose. Theprotein contribution to the conjugate is usually not considered whencalculating the dose of a conjugate. Generally, each dose will comprise0.1 to 100 mcg of polysaccharide, particularly 0.1 to 10 mcg, and moreparticularly 1 to 10 mcg.

One embodiment of the disclosure provides an immunogenic compositioncomprising any of the glycoconjugates comprising a S. pneumoniaecapsular polysaccharide conjugated to a carrier protein described above.

The immunogenic compositions of the present disclosure can be used toprotect or treat a human susceptible to bacterial infection, e.g., by aS. pneumoniae bacteria or a N. meningitidis bacteria, by means ofadministering the immunogenic compositions via a systemic, dermal ormucosal route, or can be used to generate a polyclonal or monoclonalantibody preparation that could be used to confer passive immunity onanother subject. These administrations can include injection via theintramuscular, intraperitoneal, intradermal or subcutaneous routes; orvia mucosal administration to the oral/alimentary, respiratory orgenitourinary tracts. Immunogenic compositions may also be used togenerate antibodies that are functional as measured by the killing ofbacteria in either an animal efficacy model or via an opsonophagocytickilling assay.

Optimal amounts of components for a particular immunogenic compositioncan be ascertained by standard studies involving observation ofappropriate immune responses in subjects. Following an initialvaccination, subjects can receive one or several booster immunizationsadequately spaced.

In one embodiment, the immunogenic compositions of the disclosurefurther comprise at least one of an adjuvant, a buffer, acryoprotectant, a salt, a divalent cation, a non-ionic detergent, aninhibitor of free radical oxidation, a diluent or a carrier. In oneembodiment, the adjuvant within the immunogenic composition of thedisclosure is an aluminum-based adjuvant. In one embodiment, theadjuvant is an aluminum-based adjuvant selected from the groupconsisting of aluminum phosphate, aluminum sulfate and aluminumhydroxide. In one embodiment, the adjuvant is aluminum phosphate.

An adjuvant is a substance that enhances the immune response whenadministered together with an immunogen or antigen. A number ofcytokines or lymphokines have been shown to have immune modulatingactivity, and thus may be useful in a manner the same or similar toadjuvants, including, but not limited to, the interleukins 1-α, 1-β, 2,4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15,16, 17 and 18 (and its mutant forms); the interferons-α, β and γ;granulocyte-macrophage colony stimulating factor (GM-CSF) (see, e.g.,U.S. Pat. No. 5,078,996 and ATCC Accession Number 39900); macrophagecolony stimulating factor (M-CSF); granulocyte colony stimulating factor(G-CSF); and the tumor necrosis factors α and β. Still other adjuvantsthat are useful with the immunogenic compositions described hereininclude chemokines, including without limitation, MCP-1, MIP-1α, MIP-1β,and RANTES; adhesion molecules, such as a selectin, e.g., L-selectin,P-selectin and E-selectin; mucin-like molecules, e.g., CD34, GlyCAM-1and MadCAM-1; a member of the integrin family such as LFA-1, VLA-1,Mac-1 and p150.95; a member of the immunoglobulin superfamily such asPECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3;co-stimulatory molecules such as B7-1, B7-2, CD40 and CD40L; growthfactors including vascular growth factor, nerve growth factor,fibroblast growth factor, epidermal growth factor, PDGF, BL-1, andvascular endothelial growth factor; receptor molecules including Fas,TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD,NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6; and Caspases,including ICE.

Suitable adjuvants used to enhance an immune response may furtherinclude, without limitation, MPL™ (3-O-deacylated monophosphoryl lipidA, Corixa; Hamilton, Mont.), which is described in U.S. Pat. No.4,912,094. Also suitable for use as adjuvants are synthetic lipid Aanalogs or aminoalkyl glucosamine phosphate compounds (AGP), orderivatives or analogs thereof, which are available from Corixa, andthose that are described in U.S. Pat. No. 6,113,918. One such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529). This 529 adjuvantis formulated as an aqueous form (AF) or as a stable emulsion (SE).

Still other adjuvants include muramyl peptides, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE);oil-in-water emulsions, such as MF59 (U.S. Pat. No. 6,299,884)(containing 5% Squalene, 0.5% polysorbate 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.)), and SAF (containing 10%Squalene, 0.4% polysorbate 80, 5% pluronic-blocked polymer L121, andthr-MDP, either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion); incomplete Freund's adjuvant(IFA); aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate; Amphigen; Avridine; L121/squalene;D-lactide-polylactide/glycoside; pluronic polyols; killed Bordetella;saponins, such as STIMULON™ QS-21 (Antigenics, Framingham, Mass.),described in U.S. Pat. No. 5,057,540, ISCOMATRIX™ (CSL Limited,Parkville, Australia), described in U.S. Pat. No. 5,254,339, andimmunostimulating complexes (ISCOMS); Mycobacterium tuberculosis;bacterial lipopolysaccharides; synthetic polynucleotides such asoligonucleotides containing a CpG motif (e.g., U.S. Pat. No. 6,207,646);IC-31 (Intercell AG, Vienna, Austria), described in EP Patent Nos.1,296,713 and 1,326,634; a pertussis toxin (PT) or mutant thereof, acholera toxin or mutant thereof (e.g., U.S. Pat. Nos. 7,285,281,7,332,174, 7,361,355 and 7,384,640); or an E. coli heat-labile toxin(LT) or mutant thereof, particularly LT-K63, LT-R72 (e.g., U.S. Pat.Nos. 6,149,919, 7,115,730 and 7,291,588).

The immunogenic composition optionally can comprise a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers includecarriers approved by a regulatory agency of a Federal, a stategovernment, or other regulatory agency, or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans as well as non-human mammals. The term carriermay be used to refer to a diluent, adjuvant, excipient, or vehicle withwhich the pharmaceutical composition is administered. Water, salinesolutions and aqueous dextrose and glycerol solutions can be employed asliquid carriers, particularly for injectable solutions. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulation should suitthe mode of administration.

The immunogenic compositions of the present disclosure can furthercomprise one or more additional immunomodulators, which are agents thatperturb or alter the immune system, such that either up-regulation ordown-regulation of humoral and/or cell-mediated immunity is observed. Inone embodiment, up-regulation of the humoral and/or cell-mediated armsof the immune system is provided.

Examples of certain immunomodulators include, e.g., an adjuvant orcytokine, or ISCOMATRIX™ (CSL Limited; Parkville, Australia), describedin U.S. Pat. No. 5,254,339 among others. Non-limiting examples ofadjuvants that can be used in the immunogenic composition of the presentdisclosure include the RIBI adjuvant system (Ribi Inc.; Hamilton,Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-wateremulsions, water-in-oil emulsions such as, e.g., Freund's complete andincomplete adjuvants, Block copolymer (CytRx; Atlanta, Ga.), QS-21(Cambridge Biotech Inc.; Cambridge, Mass.), SAF-M (Chiron; Emeryville,Calif.), AMPHIGEN™ adjuvant, saponin, Quil A or other saponin fraction,monophosphoryl lipid A, and avridine(N,N-Dioctadecyl-N′,N′-bis(2-hydroxyethyl)-1,3-diaminopropane,N,N-Dioctadecyl-N′,N′-bis(2-hydroxyethyl)propanediamine) lipid-amineadjuvant. Non-limiting examples of oil-in-water emulsions useful in theimmunogenic composition of the disclosure include modified SEAM62 andSEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsioncontaining 5% (v/v) squalene (Sigma), 1% (v/v) SPAN™ 85 detergent (ICISurfactants), 0.7% (v/v) polysorbate 80 detergent (ICI Surfactants),2.5% (v/v) ethanol, 200 mcg/mL Quil A, 100 mcg/ml cholesterol, and 0.5%(v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising5% (v/v) squalene, 1% (v/v) SPAN™ 85 detergent, 0.7% (v/v) polysorbate80 detergent, 2.5% (v/v) ethanol, 100 mcg/ml Quil A, and 50 mcg/mlcholesterol. Other immunomodulators that can be included in theimmunogenic composition include, e.g., one or more interleukins,interferons, or other known cytokines or chemokines. In one embodiment,the adjuvant may be a cyclodextrin derivative or a polyanionic polymer,such as those described in U.S. Pat. Nos. 6,165,995 and 6,610,310,respectively. It is to be understood that the immunomodulator and/oradjuvant to be used will depend on the subject to which the immunogeniccomposition will be administered, the route of injection and the numberof injections to be given.

The immunogenic compositions of the disclosure may further comprise oneor more preservatives in addition to a plurality of capsularpolysaccharide-protein conjugates. The FDA requires that biologicalproducts in multiple-dose (multi-dose) vials contain a preservative,with only a few exceptions. Vaccine products containing preservativesinclude vaccines containing benzethonium chloride (anthrax),2-phenoxyethanol (DTaP, HepA, Lyme, Polio (parenteral)), phenol (Pneumo,Typhoid (parenteral), Vaccinia) and thimerosal (DTaP, DT, Td, HepB, Hib,Influenza, JE, Mening, Pneumo, Rabies). Preservatives approved for usein injectable drugs include, e.g., chlorobutanol, m-cresol,methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride,benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosaland phenylmercuric nitrate.

Packaging and Dosing Forms

Formulations of the disclosure may further comprise one or more of abuffer, a salt, a divalent cation, a non-ionic detergent, acryoprotectant such as a sugar, and an anti-oxidant such as a freeradical scavenger or chelating agent, or any multiple combinationthereof. The choice of any one component, e.g., a chelator, maydetermine whether or not another component (e.g., a scavenger) isdesirable. The final composition formulated for administration should besterile and/or pyrogen free. The skilled artisan may empiricallydetermine which combinations of these and other components will beoptimal for inclusion in the preservative containing immunogeniccompositions of the disclosure depending on a variety of factors such asthe particular storage and administration conditions required.

In certain embodiments, a formulation of the disclosure which iscompatible with parenteral administration comprises one or morephysiologically acceptable buffers selected from, but not limited to,Tris (trimethamine), phosphate, acetate, borate, citrate, glycine,histidine and succinate. In certain embodiments, the formulation isbuffered to within a pH range of about 6.0 to about 9.0, preferably fromabout 6.4 to about 7.4.

In certain embodiments, it may be desirable to adjust the pH of theimmunogenic composition or formulation of the disclosure. The pH of aformulation of the disclosure may be adjusted using standard techniquesin the art. The pH of the formulation may be adjusted to be between 3.0and 8.0. In certain embodiments, the pH of the formulation may be, ormay adjusted to be, between 3.0 and 6.0, 4.0 and 6.0, or 5.0 and 8.0. Inother embodiments, the pH of the formulation may be, or may adjusted tobe, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5,about 5.8, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. Incertain embodiments, the pH may be, or may adjusted to be, in a rangefrom 4.5 to 7.5, or from 4.5 to 6.5, from 5.0 to 5.4, from 5.4 to 5.5,from 5.5 to 5.6, from 5.6 to 5.7, from 5.7 to 5.8, from 5.8 to 5.9, from5.9 to 6.0, from 6.0 to 6.1, from 6.1 to 6.2, from 6.2 to 6.3, from 6.3to 6.5, from 6.5 to 7.0, from 7.0 to 7.5 or from 7.5 to 8.0. In aspecific embodiment, the pH of the formulation is about 5.8.

In certain embodiments, a formulation of the disclosure which iscompatible with parenteral administration comprises one or more divalentcations, including but not limited to MgCl₂, CaCl₂ and MnCl₂, at aconcentration ranging from about 0.1 mM to about 10 mM, with up to about5 mM being preferred.

In certain embodiments, a formulation of the disclosure which iscompatible with parenteral administration comprises one or more salts,including but not limited to sodium chloride, potassium chloride, sodiumsulfate, and potassium sulfate, present at an ionic strength which isphysiologically acceptable to the subject upon parenteral administrationand included at a final concentration to produce a selected ionicstrength or osmolarity in the final formulation. The final ionicstrength or osmolality of the formulation will be determined by multiplecomponents (e.g., ions from buffering compound(s) and othernon-buffering salts. A preferred salt, NaCl, is present from a range ofup to about 250 mM, with salt concentrations being selected tocomplement other components (e.g., sugars) so that the final totalosmolarity of the formulation is compatible with parenteraladministration (e.g., intramuscular or subcutaneous injection) and willpromote long term stability of the immunogenic components of theimmunogenic composition formulation over various temperature ranges.Salt-free formulations will tolerate increased ranges of the one or moreselected cryoprotectants to maintain desired final osmolarity levels.

In certain embodiments, a formulation of the disclosure which iscompatible with parenteral administration comprises one or morecryoprotectants selected from but not limited to disaccharides (e.g.,lactose, maltose, sucrose or trehalose) and polyhydroxy hydrocarbons(e.g., dulcitol, glycerol, mannitol and sorbitol).

In certain embodiments, the osmolarity of the formulation is in a rangeof from about 200 mOs/L to about 800 mOs/L, with a preferred range offrom about 250 mOs/L to about 500 mOs/L, or about 300 mOs/L-about 400mOs/L. A salt-free formulation may contain, for example, from about 5%to about 25% sucrose, and preferably from about 7% to about 15%, orabout 10% to about 12% sucrose. Alternatively, a salt-free formulationmay contain, for example, from about 3% to about 12% sorbitol, andpreferably from about 4% to 7%, or about 5% to about 6% sorbitol. Ifsalt such as sodium chloride is added, then the effective range ofsucrose or sorbitol is relatively decreased. These and other suchosmolality and osmolarity considerations are well within the skill ofthe art.

In certain embodiments, a formulation of the disclosure which iscompatible with parenteral administration comprises one or more freeradical oxidation inhibitors and/or chelating agents. A variety of freeradical scavengers and chelators are known in the art and apply to theformulations and methods of use described herein. Examples include butare not limited to ethanol, EDTA, an EDTA/ethanol combination,triethanolamine, mannitol, histidine, glycerol, sodium citrate, inositolhexaphosphate, tripolyphosphate, ascorbic acid/ascorbate, succinicacid/succinate, malic acid/maleate, desferal, EDDHA and DTPA, andvarious combinations of two or more of the above. In certainembodiments, at least one non-reducing free radical scavenger may beadded at a concentration that effectively enhances long term stabilityof the formulation. One or more free radical oxidationinhibitors/chelators may also be added in various combinations, such asa scavenger and a divalent cation. The choice of chelator will determinewhether or not the addition of a scavenger is needed.

In certain embodiments, a formulation of the disclosure which iscompatible with parenteral administration comprises one or morenon-ionic surfactants, including but not limited to polyoxyethylenesorbitan fatty acid esters, polysorbate-80 (TWEEN™ 80), polysorbate-60(TWEEN™ 60), polysorbate-40 (TWEEN™ 40) and polysorbate-20 (TWEEN™ 20),polyoxyethylene alkyl ethers, including but not limited to Brij 58, Brij35, as well as others such as TRITON™ X-100; TRITON™ X-114, NP40 (nonylphenoxypolyethoxylethanol), SPAN™ 85 and the PLURONIC™ series ofnon-ionic surfactants (e.g., PLURONIC™ 121), with preferred componentspolysorbate-80 at a concentration from about 0.001% to about 2% (with upto about 0.25% being preferred) or polysorbate-40 at a concentrationfrom about 0.001% to 1% (with up to about 0.5% being preferred).

In certain embodiments, a formulation of the disclosure comprises one ormore additional stabilizing agents suitable for parenteraladministration, e.g., a reducing agent comprising at least one thiol(—SH) group (e.g., cysteine, N-acetyl cysteine, reduced glutathione,sodium thioglycolate, thiosulfate, monothioglycerol, or mixturesthereof). Alternatively or optionally, preservative-containingimmunogenic composition formulations of the disclosure may be furtherstabilized by removing oxygen from storage containers, protecting theformulation from light (e.g., by using amber glass containers).

Preservative-containing immunogenic composition formulations of thedisclosure may comprise one or more pharmaceutically acceptable carriersor excipients, which include any excipient that does not itself inducean immune response. Suitable excipients include but are not limited tomacromolecules such as proteins, saccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers,sucrose (Paoletti et al, 2001, Vaccine, 19:2118), trehalose, lactose andlipid aggregates (such as oil droplets or liposomes). Such carriers arewell known to the skilled artisan. Pharmaceutically acceptableexcipients are discussed, e.g., in Gennaro, 2000, Remington: The Scienceand Practice of Pharmacy, 20^(th) edition, ISBN:0683306472.

Compositions of the disclosure may be lyophilized or in aqueous form,i.e. solutions or suspensions. Liquid formulations may advantageously beadministered directly from their packaged form and are thus ideal forinjection without the need for reconstitution in aqueous medium asotherwise required for lyophilized compositions of the disclosure.

Direct delivery of immunogenic compositions of the present disclosure toa subject may be accomplished by parenteral administration(intramuscularly, intraperitoneally, intradermally, subcutaneously,intravenously, or to the interstitial space of a tissue); or by rectal,oral, vaginal, topical, transdermal, intranasal, ocular, aural,pulmonary or other mucosal administration. In a preferred embodiment,parenteral administration is by intramuscular injection, e.g., to thethigh or upper arm of the subject. Injection may be via a needle (e.g. ahypodermic needle), but needle free injection may alternatively be used.A typical intramuscular dose is 0.5 mL. Compositions of the disclosuremay be prepared in various forms, e.g., for injection either as liquidsolutions or suspensions. In certain embodiments, the composition may beprepared as a powder or spray for pulmonary administration, e.g. in aninhaler. In other embodiments, the composition may be prepared as asuppository or pessary, or for nasal, aural or ocular administration,e.g. as a spray, drops, gel or powder. Optimal amounts of components fora particular immunogenic composition may be ascertained by standardstudies involving observation of appropriate immune responses insubjects. Following an initial vaccination, subjects can receive one orseveral booster immunizations adequately spaced.

Immunogenic compositions of the disclosure may be packaged in unit doseor multi-dose form (e.g. 2 doses, 4 doses, or more). For multi-doseforms, vials are typically but not necessarily preferred over pre-filledsyringes. Suitable multi-dose formats include but are not limited to: 2to 10 doses per container at 0.1 to 2 mL per dose. In certainembodiments, the dose is a 0.5 mL dose. See, e.g., International PatentApplication WO2007/127668, which is incorporated by reference herein.

Compositions may be presented in vials or other suitable storagecontainers, or may be presented in pre-filled delivery devices, e.g.,single or multiple component syringes, which may be supplied with orwithout needles. A syringe typically but need not necessarily contains asingle dose of the preservative-containing immunogenic composition ofthe disclosure, although multi-dose, pre-filled syringes are alsoenvisioned. Likewise, a vial may include a single dose but mayalternatively include multiple doses.

Effective dosage volumes can be routinely established, but a typicaldose of the composition for injection has a volume of 0.5 mL. In certainembodiments, the dose is formulated for administration to a humansubject. In certain embodiments, the dose is formulated foradministration to an adult, teen, adolescent, toddler or infant (i.e.,no more than one year old) human subject and may in preferredembodiments be administered by injection.

Liquid immunogenic compositions of the disclosure are also suitable forreconstituting other immunogenic compositions which are presented inlyophilized form. Where an immunogenic composition is to be used forsuch extemporaneous reconstitution, the disclosure provides a kit withtwo or more vials, two or more ready-filled syringes, or one or more ofeach, with the contents of the syringe being used to reconstitute thecontents of the vial prior to injection, or vice versa.

Alternatively, immunogenic compositions of the present disclosure may belyophilized and reconstituted, e.g., using one of a multitude of methodsfor freeze drying well known in the art to form dry, regular shaped(e.g., spherical) particles, such as micropellets or microspheres,having particle characteristics such as mean diameter sizes that may beselected and controlled by varying the exact methods used to preparethem. The immunogenic compositions may further comprise an adjuvantwhich may optionally be prepared with or contained in separate dry,regular shaped (e.g., spherical) particles such as micropellets ormicrospheres. In such embodiments, the present disclosure furtherprovides an immunogenic composition kit comprising a first componentthat includes a stabilized, dry immunogenic composition, optionallyfurther comprising one or more preservatives of the disclosure, and asecond component comprising a sterile, aqueous solution forreconstitution of the first component. In certain embodiments, theaqueous solution comprises one or more preservatives, and may optionallycomprise at least one adjuvant (see, e.g., WO2009/109550 (incorporatedherein by reference)).

In yet another embodiment, a container of the multi-dose format isselected from one or more of the group consisting of, but not limitedto, general laboratory glassware, flasks, beakers, graduated cylinders,fermentors, bioreactors, tubings, pipes, bags, jars, vials, vialclosures (e.g., a rubber stopper, a screw on cap), ampoules, syringes,dual or multi-chamber syringes, syringe stoppers, syringe plungers,rubber closures, plastic closures, glass closures, cartridges anddisposable pens and the like. The container of the present disclosure isnot limited by material of manufacture, and includes materials such asglass, metals (e.g., steel, stainless steel, aluminum, etc.) andpolymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).In a particular embodiment, the container of the format is a 5 mL SchottType 1 glass vial with a butyl stopper. The skilled artisan willappreciate that the format set forth above is by no means an exhaustivelist, but merely serve as guidance to the artisan with respect to thevariety of formats available for the present disclosure. Additionalformats contemplated for use in the present disclosure may be found inpublished catalogues from laboratory equipment vendors and manufacturerssuch as United States Plastic Corp. (Lima, Ohio), VWR.

Methods for Inducing an Immune Response and Protecting Against Infection

The present disclosure also includes methods of use for immunogeniccompositions described herein. For example, one embodiment of thedisclosure provides a method of inducing an immune response against apathogenic bacteria, for example S. pneumonia, comprising administeringto a subject an immunogenic amount of any of the immunogeniccompositions described herein comprising a bacterial antigen such as abacterial capsular polysaccharide derived from pathogenic bacteria. Oneembodiment of the disclosure provides a method of protecting a subjectagainst an infection with S. pneumoniae, or a method of preventinginfection with S. pneumoniae, or a method of reducing the severity of ordelaying the onset of at least one symptom associated with an infectioncaused by S. pneumoniae, the methods comprising administering to asubject an immunogenic amount of any of the immunogenic compositionsdescribed herein comprising a bacterial antigen such as a bacterialcapsular polysaccharide derived from S. pneumoniae. One embodiment ofthe disclosure provides a method of treating or preventing aStreptococcal infection, disease or condition associated with aStreptococcus sp. in a subject, the method comprising the step ofadministering a therapeutically or prophylactically effective amount ofan immunogenic composition described herein to the subject. In someembodiments, the method of treating or preventing a Streptococcalinfection, disease or conditions comprises human, veterinary, animal, oragricultural treatment. Another embodiment provides a method of treatingor preventing a Streptococcal infection, disease or condition associatedwith a Streptococcus sp. in a subject, the method comprising generatinga polyclonal or monoclonal antibody preparation from the immunogeniccomposition described herein, and using said antibody preparation toconfer passive immunity to the subject. One embodiment of the disclosureprovides a method of preventing a Streptococcal infection in a subjectundergoing a surgical procedure, the method comprising the step ofadministering a prophylactically effective amount of an immunogeniccomposition described herein to the subject prior to the surgicalprocedure.

An “immune response” to an antigen or immunogenic composition is thedevelopment in a subject of a humoral and/or a cell-mediated immuneresponse to molecules present in the antigen or vaccine composition ofinterest. For purposes of the present disclosure, a “humoral immuneresponse” is an antibody-mediated immune response and involves theinduction and generation of antibodies that recognize and bind with someaffinity for the antigen in the immunogenic composition of thedisclosure, while a “cell-mediated immune response” is one mediated byT-cells and/or other white blood cells. A “cell-mediated immuneresponse” is elicited by the presentation of antigenic epitopes inassociation with Class I or Class II molecules of the majorhistocompatibility complex (MHC), CD1 or other non-classical MHC-likemolecules. This activates antigen-specific CD4+ T helper cells or CD8+cytotoxic T lymphocyte cells (“CTLs”). CTLs have specificity for peptideantigens that are presented in association with proteins encoded byclassical or non-classical MHCs and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide or other antigens in associationwith classical or non-classical MHC molecules on their surface. A“cell-mediated immune response” also refers to the production ofcytokines, chemokines and other such molecules produced by activatedT-cells and/or other white blood cells, including those derived fromCD4+ and CD8+ T-cells. The ability of a particular antigen orcomposition to stimulate a cell-mediated immunological response may bedetermined by a number of assays, such as by lymphoproliferation(lymphocyte activation) assays, CTL cytotoxic cell assays, by assayingfor T-lymphocytes specific for the antigen in a sensitized subject, orby measurement of cytokine production by T cells in response tore-stimulation with antigen. Such assays are well known in the art. See,e.g., Erickson et al. (1993) J. Immunol. 151:4189-4199; and Doe et al.(1994) Eur. J. Immunol. 24:2369-2376.

As used herein, “treatment” (including variations thereof, e.g., “treat”or “treated”) means any one or more of the following: (i) the preventionof infection or re-infection, as in a traditional vaccine, (ii) thereduction in the severity of, or, in the elimination of symptoms, and(iii) the substantial or complete elimination of the pathogen ordisorder in question. Hence, treatment may be effected prophylactically(prior to infection) or therapeutically (following infection). In thepresent disclosure, prophylactic treatment is the preferred mode.According to a particular embodiment of the present disclosure,compositions and methods are provided that treat, includingprophylactically and/or therapeutically immunize, a host animal againsta microbial infection (e.g., a bacterium such as Streptococcus). Themethods of the present disclosure are useful for conferring prophylacticand/or therapeutic immunity to a subject. The methods of the presentdisclosure can also be practiced on subjects for biomedical researchapplications.

As used herein, “mammal” means a human or non-human animal. Moreparticularly, mammal refers to any animal classified as a mammal,including humans, domestic and farm animals, and research, zoo, sportsand pet companion animals such as a household pet and other domesticatedanimal including, but not limited to, cattle, sheep, ferrets, swine,horses, rabbits, goats, dogs, cats, and the like. Preferred companionanimals are dogs and cats. Preferably, the mammal is human.

An “immunogenic amount,” and “immunologically effective amount,” both ofwhich are used interchangeably herein, refers to the amount of antigenor immunogenic composition sufficient to elicit an immune response,either a cellular (T-cell) or humoral (B-cell or antibody) response, orboth, as measured by standard assays known to one skilled in the art.

The amount of a particular conjugate in a composition is generallycalculated based on total polysaccharide, conjugated and non-conjugatedfor that conjugate. For example, a conjugate with 20% freepolysaccharide will have about 80 mcg of conjugated polysaccharide andabout 20 mcg of non-conjugated polysaccharide in a 100 mcgpolysaccharide dose. The protein contribution to the conjugate isusually not considered when calculating the dose of a conjugate. Theamount of conjugate can vary depending upon the streptococcal serotype.Generally, each dose will comprise 0.1 to 100 mcg of polysaccharide,particularly 0.1 to 10 mcg, and more particularly 1 to 10 mcg. The“immunogenic amount” of the different polysaccharide components in theimmunogenic composition, may diverge and each may comprise 1 mcg, 2 mcg,3 mcg, 4 mcg, 5 mcg, 6 mcg, 7 mcg, 8 mcg, 9 mcg, 10 mcg, 15 mcg, 20 mcg,30 mcg, 40 mcg, 50 mcg, 60 mcg, 70 mcg, 80 mcg, 90 mcg, or about 100 mcgof any particular polysaccharide antigen.

S. pneumoniae “invasive disease” is the isolation of bacteria from anormally sterile site, where there is associated clinical signs/symptomsof disease. Normally sterile body sites include blood, CSF, pleuralfluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone,internal body site (lymph node, brain, heart, liver, spleen, vitreousfluid, kidney, pancreas, ovary) or other normally sterile sites.Clinical conditions characterizing invasive diseases include bacteremia,pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock andmore.

The effectiveness of an antigen as an immunogen, can be measured eitherby proliferation assays, by cytolytic assays, such as chromium releaseassays to measure the ability of a T-cell to lyse its specific targetcell, or by measuring the levels of B-cell activity by measuring thelevels of circulating antibodies specific for the antigen in serum. Animmune response may also be detected by measuring the serum levels ofantigen specific antibody induced following administration of theantigen, and more specifically, by measuring the ability of theantibodies so induced to enhance the opsonophagocytic ability ofparticular white blood cells, as described herein. The level ofprotection of the immune response may be measured by challenging theimmunized host with the antigen that has been administered. For example,if the antigen to which an immune response is desired is a bacterium,the level of protection induced by the immunogenic amount of the antigenis measured by detecting the percent survival or the percent mortalityafter challenge of the animals with the bacterial cells. In oneembodiment, the amount of protection may be measured by measuring atleast one symptom associated with the bacterial infection, e.g., a feverassociated with the infection. The amount of each of the antigens in themulti-antigen or multi-component vaccine or immunogenic compositionswill vary with respect to each of the other components and can bedetermined by methods known to the skilled artisan. Such methods wouldinclude procedures for measuring immunogenicity and/or in vivo efficacy.In certain embodiments, the term “about” means within 20%, preferablywithin 10%, and more preferably within 5% of the indicated value orrange

The disclosure further provides antibodies and antibody compositionswhich bind specifically and selectively to the capsular polysaccharidesor glycoconjugates of the present disclosure. In some embodiments,antibodies are generated upon administration to a subject of thecapsular polysaccharides or glycoconjugates of the present disclosure.In some embodiments, the disclosure provides purified or isolatedantibodies directed against one or more of the capsular polysaccharidesor glycoconjugates of the present disclosure. In some embodiments, theantibodies of the present disclosure are functional as measured bykilling bacteria in either an animal efficacy model or via anopsonophagocytic killing assay. In some embodiments, the antibodies ofthe disclosure confer passive immunity to a subject. The presentdisclosure further provides polynucleotide molecules encoding anantibody or antibody fragment of the disclosure, and a cell, cell line(such as hybridoma cells or other engineered cell lines for recombinantproduction of antibodies) or a transgenic animal that produces anantibody or antibody composition of the disclosure, using techniqueswell-known to those of skill in the art.

Antibodies or antibody compositions of the disclosure may be used in amethod of treating or preventing a Staphylococcal infection, disease orcondition associated with a Streptococcus sp. in a subject, the methodcomprising generating a polyclonal or monoclonal antibody preparation,and using said antibody or antibody composition to confer passiveimmunity to the subject. Antibodies of the disclosure may also be usefulfor diagnostic methods, e.g., detecting the presence of or quantifyingthe levels of capsular polysaccharide or a glycoconjugate thereof.

The following examples are provided by way of illustration and not byway of limitation. Abbreviations: MW=molecular weight; WFI=water forinjection; TEMPO=2,2,6,6-Tetramethyl-1-piperidinyloxy free radical;NCS=N-Chlorosuccinimide.

EXAMPLES Example 1 Conjugation of Pn Serotype-12F Using TEMPO/NCS

In order to improve the stability of Serotype 12F-CRM₁₉₇glycoconjugates, alternate chemistries were explored using2,2,6,6-Tetramethyl-1-piperidinyloxy free radical (TEMPO) andN-Chlorosuccinimide (NCS) as the cooxidant to oxidize primary alcoholsto aldehyde groups. GC/MS analysis showed that the sites of oxidationwere different from that of periodate mediated oxidation. In the case ofTEMPO-NCS oxidation, the α-D-Glcp and 2-Glcp were oxidized, whereasα-D-Galp was the major site of oxidation when periodate was used (seeFIG. 1). As described in further detail herein, TEMPO was used incatalytic amounts (≦0.1 molar equivalents) and the desired degree ofoxidation (DO) was achieved by varying the amounts of NCS used.Subsequently several conjugates were synthesized and characterized. Ingeneral, the production of Serotype 12F glycoconjugates was carried outin several phases, as follows:

1) Hydrolysis of Serotype 12 polysaccharide to molecular weights 50 to500 kDa.

2) Activation of Serotype 12F polysaccharide with TEMPO/NCS

3) Purification of the activated polysaccharide

4) Conjugation of activated Serotype 12F to CRM₁₉₇ protein

5) Purification of Serotype 12F-CRM conjugates.

Example 2 Hydrolysis and Oxidation of Serotype 12F

The hydrolysis of the polysaccharideis was typically performed underacidic conditions with heating to obtain an average molecular weight inthe desired range of 100 to 350 kDa. A typical experiment is describedbelow.

Hydrolysis

The Serotype 12F polysaccharide solution was added to a jacketedreaction vessel. To this, the required volume of 0.30 M Acetic acid andwater for injection (WFI) were added to maintain ˜0.1 M acetic acidconcentration. The pH of the solution was adjusted to 3.2±0.3 using 1 NNaOH or Glacial Acetic acid. The temperature of the reaction mixture wasincreased to 70±5° C. The reaction mixture was stirred at 70±5° C. for90-120 minutes. The reaction mixture was cooled down to 23±2° C. andneutralized (pH 7.0) by adding 1 M NaOH solution. The hydrolyzedpolysaccharide was purified by ultrafiltration/diafiltration against WFIusing 30K MWCO membranes. The solution was filtered through a 0.22 μmfilter and stored at 2 to 8° C. until oxidation. The molecular weight ofthe hydrolyzed polysaccharide was analyzed by SEC-MALLS to ensure thatthe molecular weight met the target range of 100 to 350 kDa.

Partial Oxidation

In one experiment, the Serotype 12F polysaccharide was mechanicallysized using pressure homogenization using a microfluidiser to reduce themolecular weight to approximately 100 to 500 kDa. The sizedpolysaccharide was added to a reaction vessel at a concentration of 4.0mg/mL and mixed with bicarbonate/carbonate buffer (0.5 M NaHCO₃/0.05 MNa₂CO₃ buffer, pH 8.6) at a ratio of 1:1 v/v. To the stirred mixture wasadded ≦0.1 mol equivalent of TEMPO. The reaction was started by theaddition of 0.6 to 1.0 mol equivalent of NCS. The reaction mixture wasstirred at room temperature for 2 hours, after which the activatedpolysaccharide was purified by diafiltration, with WFI using a 30Kultrafiltration membrane. The purified polysaccharide was collected andthe degree of oxidation (DO) was determined by quantitative measurementsof aldehyde (using a 3-methyl-2-benothiazolinone hydrazone (MBTH) assay)and polysaccharide (using an anthrone assay).

In another experiment, the Serotype 12F polysaccharide was hydrolyzed toreduce the molecular weight to a molecular weight of approximately 100to 500 kDa. The Serotype 12F polysaccharide was added to a reactionvessel and mixed with 0.5 M NaHCO₃/0.05 M Na₂CO₃ buffer (pH 8.6) at aratio of 1:1 v/v. To the stirred mixture was added 0.6 to 1.0 molarequivalents of NCS dissolved in WFI. The activation was initiated by theaddition of approximately 0.1 molar equivalents of TEMPO dissolved inWFI. The reaction mixture was stirred at room temperature for 2 hours,after which the activated polysaccharide was purified by diafiltrationwith WFI using a 30K ultrafiltration membrane. The purified activatedpolysaccharide was filtered through a 0.2 μm filter and stored at 4° C.before use.

The TEMPO/NCS mediated oxidations were also performed successfully insodium phosphate buffers of pH 6.5, 7.0, 7.5 and 8.0. In some activationexperiments a primary alcohol such as n-propanol was used to quench thereagents in order to avoid saccharide overoxidation. In another set ofexperiments the chemically hydrolysed polysaccharide was subjected tooxidation directly, without the ultrafiltration/diafiltrationpurification step.

Example 3 Conjugation of Serotype 12F Oxidized Polysaccharide

In one experiment, the purified oxidized Serotype 12F polysaccharide wasadded to a reaction vessel followed by the addition of 0.5 M Sodiumphosphate buffer (pH 6.5) to a final buffer concentration of 0.1 M. Tothis solution, previously lyophilized CRM₁₉₇ was added and mixedthoroughly in order to obtain a homogenous solution. The pH was adjustedto 6.8 using diluted HCl or 1N NaOH solution. This was followed by theaddition of 1.5 molar equivalents of NaCNBH₃. The reaction mixture wasstirred for 24 hours at room temperature (23° C.) and for 2.5 days at37° C. The reaction mixture was then diluted with 1×0.9% saline and theunreacted aldehyde groups were “capped” with 2 molar equivalents ofsodium borohydride. The capping reaction time was 3 hours.

In another experiment, the purified activated Serotype 12F was added toa reaction vessel followed by the addition of 0.5 M sodium phosphatebuffer (pH 6.5) to a final buffer concentration of 0.1 M. To thissolution, previously lyophilized CRM₁₉₇ was added and mixed thoroughlyto obtain a homogenous solution. The pH was adjusted to 6.8 usingdiluted HCl or 1N NaOH solution. This was followed by the addition of 3molar equivalents of NaCNBH₃. The reaction mixture was stirred for 24hours at 23° C. and for 48 hrs at 37° C. The reaction mixture was thendiluted with 1×0.9% saline and with stirring, the unreacted aldehydegroups were “capped” with 1 molar equivalent sodium borohydride NaBH₄.The capping reaction time was 3 hours.

In another experiment, the purified activated Serotype 12F was added toa reaction vessel and mixed with CRM₁₉₇ solution. The mixture waslyophilized and the powder was dissolved in 0.1 M sodium phosphatebuffer (pH 6.8) to a final saccharide concentration of 5 mg/mL. Ifneeded the pH was adjusted to 6.8 using diluted HCl or 1N NaOH solution.This was followed by the addition of 3 molar equivalents NaCNBH₃. Thereaction mixture was stirred for 24 hours at 23° C. and for 48 hrs at37° C. The reaction mixture was then diluted with 1×0.9% saline, theunreacted aldehyde groups were “capped” with 1 molar equivalent sodiumborohydride NaBH₄. The capping reaction time was 3 hours.

Example 4 Conjugate Purification

The capped reaction mixture was filtered using a 5 μm filter and thenpurified using 100K MWCO ultra filtration membranes. The conjugate wasfirst diafiltered using 10 mM succinate/0.9% saline, pH 6.0 buffer. Thepurified conjugate was then filtered through 0.45/0.22 μm filters toobtain the bulk conjugate.

Example 5 Degree of Oxidation

Successful oxidation of primary alcohols in the Serotype 12Fpolysaccharide was achieved using the TEMPO/NCS system. The hydrolyzedSerotype 12F polysaccharides were oxidized to varying degrees ofoxidation (DO) levels by adjusting the amount of NCS cooxidant. Theeffect on DO by varying amounts of NCS using different polysaccharidebatches and molecular weights is shown in FIG. 2. Typically 0.5-2.5Molar Equivalents of NCS was used to achieve the target Degree ofOxidation. Typically the oxidation reaction is complete in 2 hours as nosignificant change in DO was observed after 2 hours.

Several Serotytpe 12F conjugates were generated and characterized usingthe TEMPO/NCS oxidized polysaccharide. The results are summarized inTable 1. Some representative conjugates were also successfully generatedusing other Pneumococcal serotypes activated with TEMPO/NCS system. Theprocedure for the generation of conjugates for other Pneumococcalserotypes was the same as the method used for Serotype 12F. The resultsare described in Tables 2 to 4.

TABLE 1 Pneumococcal Serotype 12F-CRM₁₉₇ conjugates Conjugate Batch 12F-12F- 12F- 12F- 12F- 12F- 84A 97B 147C 171D 177-6E 181F Oxidation Time 22 4 2 2 2 (hr) Degree of 12.0 6.0 9.6 12.0 11.5 11.5 Oxidation (D.O) %Activated 80 71 70 89 86 86 Saccharide Yield Activated 137 155 170 190240 240 Saccharide MW by MALLS (kDa) Conjugation process Conjugate Lyo-Lyo- Lyo- Lyo- Lyo- Results CRM CRM CRM CRM CRM Co-Lyo Saccharide 51.676.8 53.6 76.3 65.8 40.7 yield (%) Saccharide/ 1.2 0.9 1.0 1.1 1.4 0.9Protein Ratio % Free 24 10 17 20 23 14 Saccharide Mw by SEC- 2050 30003600 1500 2400 2100 MALLS (kDa)

TABLE 2 Pneumococcal Serotype 3-CRM₁₉₇ conjugates Conjugate BatchPn3-106-1 Pn3-106-4 Polysaccharide MALLS (Mw) kDa 430 430 OxidationOxidation Time (hr) 2 2 D.O. 9.4 15 % Activated Saccharide Yield 55 65Activated Saccharide MW by 340 360 SEC-MALLS (kDa) Conjugation ConjugateResults Saccharide yield (%) 29.9 55.0 Saccharide-Protein Ratio 0.7 1.6% Free Saccharide 21.0 30.0 MW by SEC-MALLS (kDa) 2100 2600

TABLE 3 Pneumococcal Serotype 33F-CRM₁₉₇ Conjugates Conjugate Batch33F-#55 33F-#63 Polysaccharide MALLS (Mw)  128 kDa  150 kDa D.O. 20  5   Yield 92% 97% Saccharide yield (%) 44% 68% Saccharide-Protein Ratio0.54 0.68 Free Saccharide <1% 1.10%  Free Protein <1% <1% Mw bySEC-MALLS (kDa) 11160 kDa 2730 kDa

TABLE 4 Pneumococcal Serotype 10A Conjugates Conjugate Batch 10A-#7710A-#78 10A-#85 10A-#88 10A-#89 10A-#103 10A-#104 Polysaccharide MALLS(Mw) 538 Kda 538 Kda 538 Kda 538 Kda 538 Kda 509 Kda 509 Kda D.O. 7.9 2412 6.9 10 11.3 5.7 Yield 82% 90% 94% 88% 94% 94% 86% Saccharide yield(%) 35 20 42 35 41 43 36 Saccharide-Protein Ratio 0.53 0.33 0.73 0.70.95 0.6 0.45 Free Saccharide <1 20 4.6 1.6 5.7 <1 <1 Free Protein <1%<1% <1% <1% <1% <1% <1% Mw by SEC-MALLS 3168 16390 4117 3137 2855 43803772

Example 6 Immunogenicity of Pn-Serotype 12F-CRM197 Conjugates Using theTEMPO/NCS Oxidation Method

The opsonophagocytic activity (OPA) titers for Serotype 12F-CRM₁₉₇conjugates in mice were determined in mice under standard conditions.OPA titers (geometric mean titer (GMT) with 95% confidence interval(CI)) at four and seven weeks are shown in Table 5, demonstrating thatthe serotype 12F-CRM₁₉₇ conjugate (Batch 12F-97B; also see Table 1 forcharacterization data of this conjugate) elicited OPA titers in a murineimmunogenicity model. The conjugate generated by the TEMPO-NCS was moreimmunogenic than the control conjugate (171B) generated from theperiodate oxidation.

TABLE 5 Immunogenicity of Serotype 12F-CRM197 Conjugates Dose ConjugateSample 0.001 ug 0.01 ug 0.1 ug Periodate Oxidation 4 16 172 (171B)Control TEMPO/NCS Oxidation 40 417 880 (12F-97B)

Example 7 Putative Mechanism for the Pn-Serotype 12F Conjugate UsingNitroxyl Radical in the Presence of an Oxidant Such as TEMPO/NCS

The putative mechanism of oxidation/conjugation of Pn-serotype 12F isshown in FIG. 6. The primary hydroxyl groups of the polysaccharide areoxidized by catalytic amounts of nitroxyl radical such as TEMPO, with anoxidant such as NCS as the stoichiometric oxidant. The actual oxidant isthe N-oxoammonium salt, in a catalytic cycle. The oxidation of the C-6primary hydroxyl groups generates aldehyde groups which are subsequentlyreacted with the primary amino groups of the lysine of the carrierprotein (CRM₁₉₇) to generate the glycoconjugate.

Example 8 Stability Comparison

Comparison of the stability (at 25° C.) of the conjugates generated byperiodate oxidation vs TEMPO/NCS oxidation (see FIG. 7) demonstratedthat the conjugate generated by the oxidation of the Pn-12Fpolysaccharides were relatively more stable. As shown in FIG. 7, anincrease in the free saccharide over time was observed for theglycoconjugate generated by the periodate oxidation of the Pn-12Fpolysaccharide at 25° C. In contrast, the glycoconjugate prepared usingthe TEMPO/NCS oxidation of the Pn-12F polysaccharide showed nosignificant trends for the free saccharide under similar conditions.

1. A method of making a glycoconjugate comprising a saccharideconjugated to a carrier protein, comprising the steps of: a) reacting asaccharide with a stable nitroxyl radical compound and an oxidant toproduce an activated saccharide; and b) reacting the activatedsaccharide with a carrier protein comprising one or more amine groups.2.-3. (canceled)
 4. The method of claim 1, wherein said stable nitroxylradical compound is a molecule bearing a TEMPO or a PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety, having the ability toselectively oxidize primary alcohols in the presence of an oxidant, togenerate aldehyde groups without affecting secondary hydroxyl groups. 5.(canceled)
 6. The method of claim 1, wherein said nitroxyl radicalcompound is selected from the group consisting of TEMPO,2,2,6,6-Tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy,4-Phosphonooxy-TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO,4-Isothiocyanato-TEMPO, 4-(2-Iodoacetamido)-TEMPO free radical,4-Hydroxy-TEMPO, 4-Cyano-TEMPO, 4-Carboxy-TEMPO,4-(2-Bromoacetamido)-TEMPO, 4-Amino-TEMPO, and4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. 7.-9. (canceled) 10.The method of claim 1, wherein said oxidant is selected from the groupconsisting of N-ChloroSuccinimide, N-Bromosuccinimide,N-Iodosuccinimide, Dichloroisocyanuric acid,1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, Dibromoisocyanuric acid,1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, Diiodoisocyanuric acid and1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. 11.-18. (canceled)
 19. Amethod of making a glycoconjugate comprising a saccharide conjugated toa carrier protein, comprising the steps of: a) reacting a saccharidewith 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups.
 20. The method of claim 1,wherein the degree of oxidation of the activated saccharide ranges from3 to
 40. 21.-25. (canceled)
 26. The method of claim 19, wherein thesaccharide is a capsular polysaccharide derived from N. meningitidis.27.-28. (canceled)
 29. The method of claim 19, wherein the saccharide isa capsular polysaccharide from Group B Streptococcus (GBS). 30.-31.(canceled)
 32. The method of claim 1, wherein the carrier protein isCRM₁₉₇. 33.-36. (canceled)
 37. The method of claim 1, wherein prior tostep a), the saccharide is hydrolyzed to a molecular weight ranging from100 to 400 kDa. 38.-63. (canceled)
 64. An immunogenic compositioncomprising the glycoconjugate produced by the method of claim 1, and apharmaceutically acceptable excipient, carrier, or diluent.
 65. Theimmunogenic composition of claim 64, further comprising an additionalantigen.
 66. The immunogenic composition of claim 65, wherein theadditional antigen comprises a protein antigen or a glycoconjugate of acapsular polysaccharide derived from S. pneumonia.
 67. (canceled) 68.The immunogenic composition of claim 65, wherein the additional antigencomprises a protein antigen or a glycoconjugate of a capsularpolysaccharide derived from N. meningitidis.
 69. (canceled)
 70. Theimmunogenic composition of claim 65, wherein the additional antigencomprises a glycoconjugate of a capsular polysaccharide derived fromGroup B Streptococcus (GBS).
 71. (canceled)
 72. The immunogeniccomposition of claim 64, further comprising an adjuvant.
 73. Theimmunogenic composition of claim 72, wherein the adjuvant is analuminum-based adjuvant.
 74. (canceled)
 75. An immunogenic compositioncomprising Pn-serotype 12F conjugated to a carrier protein, wherein theamount of free Pn-serotype 12F polysaccharide in the composition is lessthan 35% after 120 days from when it was prepared. 76.-79. (canceled)80. The immunogenic composition of claim 75, further comprising anadditional antigen. 81.-100. (canceled)